A small pig SNP molecular marker combination and application thereof

Abstract

This application relates to the technical field of biological gene detection, specifically disclosing a miniature pig SNP molecular marker combinatorial system and its application. The miniature pig SNP molecular marker combinatorial system disclosed in this application includes at least one of 221 SNP molecular markers. The physical locations of these 221 SNP molecular markers are determined based on sequence alignment during 10× coverage whole-genome sequencing of miniature pigs. The site information of the 221 SNP molecular markers is shown in Table 1. The miniature pig SNP molecular marker combinatorial system provided in this application is used for detecting SNP genotyping in miniature pigs and possesses novel, stable, cost-effective, and efficient properties.

Description

Technical Field

[0001] This application relates to the technical field of biological gene detection, specifically to a combination of SNP molecular markers for miniature pigs and their applications. Background Technology

[0002] Genetic quality monitoring of laboratory animals involves using scientific methods to test the genetic consistency of inbred strains and conduct population genetic analysis of closed colonies to evaluate whether they conform to strain or population characteristics, ensuring the inherent genetic composition and biological characteristics of the breed and strain. With the development of life sciences, the technologies and methods for testing the genetic quality of laboratory animals have continuously advanced, evolving from initial morphological, chromosome, protein molecular, and immunological techniques to molecular biology gene detection. This evolution has progressed from extensive to intensive, from qualitative to quantitative, and from cumbersome to simple. These advancements have not only significantly improved the accuracy and reliability of laboratory animal genetic quality assessment, reduced workload, and shortened testing cycles, but also ensured the reliability of laboratory animal genetic quality through advancements in genetic testing technology. This plays a crucial role in the classification, breed and strain maintenance, resource preservation, development and utilization, and animal selection and breeding.

[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 bioscience and technology has placed higher demands and challenges on genetic markers. Ideal genetic markers should possess characteristics such as high density, strong genetic stability, simple genetic behavior, insensitivity to internal and external environmental influences, and ease of automated analysis. Under these conditions, DNA molecular markers have become the most ideal genetic markers. DNA molecular markers are based on DNA sequence polymorphisms. The earliest restriction fragment length polymorphisms (RFLPs) were identified as first-generation DNA molecular markers. Second-generation DNA molecular markers are microsatellite polymorphisms, mainly composed of tandem repeats of 2, 3, or 4 nucleotides. As a third-generation genetic marker, SNPs are relatively easy to identify in the genome and can be stably inherited between parents and offspring; therefore, they are widely used in clinical and research applications.

[0004] Single nucleotide polymorphism (SNP) refers to a polymorphic change in the DNA sequence caused by a variation in a single nucleotide. The widespread existence of genomic variation was first discovered in 1960. In 1978, Kan and Dozy announced that SNPs could be used to explain differences in the origins of different populations, susceptibility to various genetic diseases, and the genomic expression of physiological traits such as appearance. Following restriction fragment length polymorphism (RSL) and simple repeat sequence polymorphism (SLSB), the emergence of third-generation genetic markers, SNPs, has played a crucial role in biomedical research.

[0005] Miniature pigs belong to the class Mammalia, order Artiodactyla, family Suidae, genus Su, and species Suidae, with a chromosome number of 2n=38. There are many breeds of miniature pigs, and their growth and reproductive performance vary due to differences in genetic background, geographical environment, and other factors. Miniature pigs are highly resistant to adverse conditions and have a strong ability to adapt to poor ecological and feed conditions. They have simple feed requirements and are relatively extensive in terms of feeding. Their average lifespan in natural environments exceeds 10 years, with a lifespan of around 15 years [5]. Miniature pigs and micro pigs are distinguished by weight. Generally, pigs weighing 45-65 kg at 12 months of age are considered miniature pigs, while pigs weighing 25-35 kg at 12 months of age are considered micro pigs. Compared with rodent experimental animals and non-human primate experimental animals, although miniature pigs are not as small, fast-growing, or easy to operate as rodent experimental animals, 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 are not suitable for rodents. Food and Drug Administrations (FDAs) worldwide mandate that new drugs undergo safety and toxicity assessments on at least one non-rodent large animal (typically monkeys, dogs, or pigs) before clinical trials. While non-human primates are the most evolutionarily related to humans, sharing high homology in cognitive abilities, reproduction, and fertility, their resources are relatively scarce, leading to high experimental costs. Furthermore, the techniques for genetically modifying non-human primates are only mastered by a few laboratories, and numerous ethical issues must be considered. In contrast, miniature pigs possess abundant genetic resources and are easier to apply their treatments to a wider range of animals.

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

[0007] Therefore, it is urgent to build an internationally advanced SNP genetic quality detection method and standard technical system applicable to miniature pigs. This will not only fill the gaps in relevant domestic and international standards, but also be of great 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] To address the aforementioned technical problems, this application provides a combination of SNP molecular markers for miniature pigs and their applications.

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

[0010] To address the shortcomings of existing methods for detecting the genetic quality of miniature pigs, such as complex operation, time-consuming and labor-intensive processes, difficulty in result analysis, and unsuitability for high-throughput detection, this application aims to establish a genetic detection method for SNP loci in miniature pig germplasm resources. This system is novel, stable, cost-effective, and efficient in detecting SNP loci in miniature pigs.

[0011] Secondly, this application provides a method for screening loci of the aforementioned miniature pig SNP molecular marker combinations, comprising the following steps:

[0012] (1) Whole genome sequencing and sequencing quality control of miniature pigs: Whole genome sequencing with a coverage of 10× was performed on miniature pigs; raw reads were filtered using fastp software; paired ends were mapped to MunDraft using BWA software; SNPs were called and filtered 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.

[0013] (2) Locus screening should be carried out according to the following five criteria: SNP loci should be screened from different genomic fragments; the genotype frequency of SNP loci should be between 25% and 75%; there should be no complete linkage between SNP loci; no other SNPs or Indels should be found in the 200bp region upstream or downstream of the selected SNP locus; and the SNP locus should be located in the missense, nonsense and discontinuity regions of the gene region.

[0014] Thirdly, this application provides a primer set for detecting the aforementioned combination of SNP molecular markers in miniature pigs, the position information of which is shown in Table 4.

[0015] Fourthly, this application provides a miniature pig SNP detection kit, which is used to detect the aforementioned miniature pig SNP molecular marker combination.

[0016] Preferably, the kit contains a primer set and / or probes for detecting the combination of molecular markers of the miniature pig SNP.

[0017] Fifthly, this application provides a miniature pig SNP gene detection system, including the miniature pig SNP molecular marker combination, the primer set of the miniature pig SNP molecular marker combination, or the miniature pig SNP detection kit.

[0018] Preferably, the miniature pig SNP gene detection system includes the following steps in sequence: collecting miniature pig samples; miniature pig DNA extraction and quality assessment; performing whole-genome resequencing of miniature pigs; comparing sequencing data, performing bioinformatics analysis, and screening candidate SNP sites located in gene regions; establishing SNP screening criteria to form a set of high-quality SNP site combinations; designing and quality verifying multiplex PCR primers for all SNP sites; developing a multi-target region capture kit for miniature pigs to genotype all SNP sites; verifying the multi-target region capture kit; and analyzing the population genetic structure.

[0019] Sixthly, this application provides the application of at least one of the aforementioned miniature pig SNP molecular marker combination, the aforementioned primer set, the aforementioned miniature pig SNP detection kit, and the aforementioned miniature pig SNP gene detection system in the SNP genetic quality detection method and standard technical system for miniature pigs.

[0020] In summary, the technical solution of this application has the following effects:

[0021] This study developed a multi-target region capture technology based on next-generation sequencing for SNP genotyping. This application discloses a miniature pig genotyping system utilizing SNP markers, and developed a multi-target region capture technology based on next-generation sequencing for SNP genotyping. Based on the next-generation sequencing results, 221 SNP loci were initially screened. Through genotyping, a multiplex PCR amplification system including 207 SNP loci was established and optimized; a reproducible screening criterion was established, and SNPs with widespread distribution and high polymorphism levels within the genome were initially screened. The detection system of this application is novel, stable, cost-effective, and efficient for SNP genotyping in miniature pigs. Attached Figure Description

[0022] Figure 1 This document outlines the specific experimental procedure for the miniature pig SNP gene detection system in this application.

[0023] Figure 2 The screening process for SNP molecular markers in this application is described.

[0024] Figure 3 This is a genetic tree diagram of the four closed populations of miniature pigs in this application.

[0025] Figure 4 Two-dimensional (A) and three-dimensional (B) principal component analysis plots of four miniature pig populations with 207 SNPs in this application. Detailed Implementation

[0026] The present application will be further described in detail below with reference to the embodiments, which should not be construed as limiting the scope of protection claimed in the present application.

[0027] Example

[0028] This embodiment provides a miniature pig SNP gene detection system, and the experimental procedure is as follows: Figure 1 As shown, the specific steps are as follows.

[0029] 1. Collection of animal specimens

[0030] Four commonly used closed herds of miniature pigs in China were selected: the Hainan Wuzhishan miniature pig closed herd (HN, 31 pigs), the Tongzhou Bama miniature pig closed herd (TZ, 29 pigs), the Harbin Veterinary Research Institute Large White pig closed herd (HSY, 27 pigs), and the Yunnan Xishuangbanna miniature pig closed herd (YNF, 30 pigs). This experiment has been approved by the Animal Experimentation and Laboratory Animal Welfare Committee of Capital Medical University (AEEI-2021-309).

[0031] 2. Preparation of genomic DNA

[0032] DNA was extracted from small pig ear tissue using the Vazyme FastPure Cell / Tissue DNA Isolation Mini Kit DC102. The absorbance (A260 / A280) values, measured using a Nanodrop 2000c micro-spectrophotometer, were between 1.8 and 2.0, indicating that the DNA passed quality control and was stored at -20°C for later use.

[0033] 3. Whole genome sequencing and sequencing quality control

[0034] This application involves 10× coverage whole-genome sequencing of 20 Hainan Wuzhishan miniature pigs and 20 Tongzhou Bama miniature pigs, aiming to identify potential polymorphic SNP sites through sequence alignment. The 10× coverage whole-genome sequencing was performed on 20 Hainan Wuzhishan miniature pigs and 20 Tongzhou Bama miniature pigs.

[0035] Raw readings were filtered using FASP software. Paired ends were mapped to MunDraft using BWA software. SNPs were retrieved and filtered using GATK4.1 software. Data quality control was performed using BCFtools software, with the following criteria set: (1) SNPs with a depth less than 2.0 were removed; (2) SNPs with a Fisherman strand depth less than 60.0 were removed; (3) SNPs with an RMS mapping quality less than 30.0 were removed; and (4) SNPs with a depth less than 8.0 were discarded.

[0036] 4. SNP locus screening

[0037] The SNP locus screening criteria in this study include the following five points:

[0038] (1) Screening for SNP loci from different genomic fragments;

[0039] (2) The genotype frequency of SNP loci is between 25% and 75%;

[0040] (3) There is no complete linkage between SNP loci;

[0041] (4) No other SNPs or Indels were found in the 200bp region upstream or downstream of the selected SNP locus;

[0042] (5) These SNP loci are located in missense, nonsense and discontinuity regions of the gene region.

[0043] Following this rigorous screening process, this study identified high-quality SNP locus combinations suitable for genetic quality assessment in miniature pigs.

[0044] Analysis of next-generation sequencing data from 40 miniature pigs identified 221 high-quality SNP loci, which will be used in the development of a miniature pig SNP detection kit. Figure 2 (Table 1).

[0045] Table 1 Information on 221 SNP loci in miniature pigs

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[0052] 5. Kit primer design and genotyping

[0053] (1) After obtaining high-quality candidate SNP loci, this application will provide the region of interest (ROI), i.e. the physical location information of the SNPs, to Beijing Aijitaikang (Jiaxing) Biotechnology Co., Ltd.

[0054] (2) Using MFEprimer v3.1 software, based on thermodynamic stability, highly specific multiplex PCR primers were designed at both ends of the ROI region; the amplified sequence length was in the range of 160bp-260bp.

[0055] (3) The quality of PCR primer pairs was verified, primer dimerization and non-specific amplification were evaluated, the designed and synthesized primers were tested, and primers with poor detection performance were replaced.

[0056] (4) In Genotyping of SNP sites was performed on the NovaSeq™ 6000 system using PE150 paired-end sequencing multi-target region capture sequencing technology.

[0057] (5) The instructions for the miniature pig SNP detection kit are as follows:

[0058] The first round of multiplex PCR reaction was as follows:

[0059] Beforehand, remove the Enhancer buffer NB(1N), Primer pool, and IGT-EM808 polymerase mixture from the -20°C freezer, place them on an ice box to thaw, briefly vortex to mix, and then centrifuge briefly. Place them on an ice box for later use.

[0060] Remove Enhancer buffer M from the 4°C freezer and allow it to thaw at room temperature.

[0061] Prepare the reaction system according to Table 2 on the ice box:

[0062] Table 2. First-round multiplex PCR reaction system

[0063] reactants Volume usage (μL) <![CDATA[ddH2O]]> 9-x Enhancer buffer NB(1N) 3.5 Enhancer buffer M 2.5 Primer pool 5 The initial sample volume was 40 ng / reaction tube. x IGT-EM808 polymerase mixture 10 total 30

[0064] The multiplex PCR reaction was performed using the following PCR instrument program: heating at 105°C; 95°C for 3 min 30 s; 98°C for 20 s; 55°C for 1 min; 60°C for 1 min; 65°C for 2 min; 72°C for 5 min; 18 cycles.

[0065] Magnetic bead purification product: Prepare 80% ethanol in advance with anhydrous ethanol and nuclease-free water, and keep it at room temperature. Please use freshly prepared 80% ethanol for magnetic bead purification whenever possible.

[0066] Remove the purified magnetic beads from the 4°C refrigerator in advance, mix them well and let them equilibrate at room temperature for 30 minutes; vortex the purified magnetic beads that have been equilibrated to room temperature and set them aside.

[0067] Add 0.9 times the volume of magnetic beads (27 μL) to a 30 μL reaction system, mix by suction or vortexing, and let stand at room temperature for 5 min.

[0068] Centrifuge briefly, place the PCR tube on a magnetic rack for 3 minutes, and wait for the solution to become clear.

[0069] Completely remove the supernatant, remove the PCR tube from the magnetic rack, add 50 μL of YF buffer B to the tube, mix well, and let stand at room temperature for 5 min.

[0070] After a brief centrifugation, place the PCR tubes on a DynaMag-96 Side magnetic rack for 3 minutes.

[0071] Keep the PCR tube on the magnetic rack, carefully remove the supernatant, add 180 μL of 80% ethanol solution to the PCR tube, and let it stand for 30 seconds.

[0072] Keep the PCR tube on the magnetic rack, discard the supernatant, add 180 μL of 80% ethanol solution to the PCR tube again, let stand for 30 seconds, and discard the supernatant.

[0073] Cap the tube and centrifuge briefly to remove any residual ethanol to the bottom. Place the PCR tube on a magnetic rack and carefully use a 10μL pipette to remove any remaining ethanol from the bottom, being careful not to pick up the magnetic beads.

[0074] Keep the PCR tube on the magnetic rack and let it stand at room temperature for 3-5 minutes to dry the magnetic beads and allow any residual ethanol to evaporate completely.

[0075] Add 24 μL of Nuclease-Free Water, remove the PCR tube from the magnetic rack, mix by pipetting or vortexing, and let stand at room temperature for 2 minutes.

[0076] Centrifuge briefly, place the PCR tube on a magnetic rack for 2 minutes, and wait for the solution to become clear.

[0077] Use a pipette to draw 13.5 μL of supernatant and transfer it to a new PCR tube. The supernatant in the tube is the purified multiplex PCR product. Label the tube and perform the PCR reaction.

[0078] The second round of adapter sequence PCR reaction was as follows:

[0079] The reaction system is prepared according to Table 3 on an ice box:

[0080] Table 3. PCR reaction system for the second round of adapter sequences

[0081] reactants Volume (μL) The multiplex PCR product purified in the previous step 13.5 Enhancer buffer M 2.5 Premixed adapter primers, 10 μM each 1 IGT-EM808 polymerase mixture 10 ddH2O 3 total 30

[0082] The reaction conditions and the PCR instrument program were as follows: heating at 105℃; 95℃ for 3 min 30 s; 98℃ for 20 s; 58℃ for 1 min; 72℃ for 30 s; 72℃ for 5 min; 9 cycles.

[0083] Second round of magnetic bead purification

[0084] Prepare 80% ethanol in advance with anhydrous ethanol and nuclease-free water, and keep it at room temperature. Please use freshly prepared 80% ethanol for magnetic bead purification whenever possible.

[0085] Remove the purified magnetic beads from the 4°C refrigerator in advance, mix them well and let them equilibrate at room temperature for 30 minutes; vortex the purified magnetic beads that have been equilibrated to room temperature and set them aside.

[0086] Add 0.9 times the volume of magnetic beads (27 μL) to a 30 μL reaction system, mix by suction or vortexing, and let stand at room temperature for 5 min.

[0087] Centrifuge briefly, place the PCR tube on a magnetic rack for 3 minutes, and wait for the solution to become clear.

[0088] Completely remove the supernatant, remove the PCR tube from the magnetic rack, add 50 μL of YF buffer B to the tube, mix well, and let stand at room temperature for 5 min.

[0089] After a brief centrifugation, place the PCR tubes on a DynaMag-96 Side magnetic rack for 3 minutes.

[0090] Keep the PCR tube on the magnetic rack, carefully remove the supernatant, add 180 μL of 80% ethanol solution to the PCR tube, and let it stand for 30 seconds.

[0091] Keep the PCR tube on the magnetic rack, discard the supernatant, add 180 μL of 80% ethanol solution to the PCR tube again, let stand for 30 seconds, and discard the supernatant.

[0092] Cap the tube and centrifuge briefly to remove any residual ethanol to the bottom. Place the PCR tube on a magnetic rack and carefully use a 10μL pipette to remove any remaining ethanol from the bottom, being careful not to pick up the magnetic beads.

[0093] Keep the PCR tube on the magnetic rack and let it stand at room temperature for 3-5 minutes to dry the magnetic beads and allow any residual ethanol to evaporate completely.

[0094] Add 24 μL of Nuclease-Free Water, remove the PCR tube from the magnetic rack, mix by pipetting or vortexing, and let stand at room temperature for 2 minutes.

[0095] Centrifuge briefly, place the PCR tube on a magnetic rack for 2 minutes, and wait for the solution to become clear.

[0096] Use a pipette to draw 20 μL of supernatant and transfer it to a new PCR tube. The supernatant in the tube contains the prepared multiplex PCR library.

[0097] Take 1 μL of the library for concentration determination and record the library concentration (the normal concentration range of the library is 20 ng / μL to 40 ng / μL, and the library concentration is mainly related to the quality of the template).

[0098] (6) The PCR primers for miniature pigs were quality-validated, and primer dimerization and non-specific amplification were evaluated. The designed and synthesized primers were tested, and primers with poor detection performance were replaced. Ultimately, the genomic matching of 221 primers reached 100% (Table 4).

[0099] Table 4 Primer locations and predicted coverage for miniature pig PCR

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[0106] 6. Validation of the multi-target region capture sequencing kit

[0107] The genetic detection kits for SNP loci in miniature pigs were simultaneously validated by three companies (Aijitaikang, Tianyi Huiyuan, and Zhongke Guobang). The sensitivity, specificity, and reproducibility of the miniature pig kits were validated as shown in Table 5 below.

[0108] Table 5. Validation of the test kit for miniature pigs

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[0110] Experimental results from three institutions showed that even at concentrations as low as 10 μg / mL, the kit could still perform multiplex PCR amplification of SNP combinations from miniature pigs and accurately genotype the SNP sites, indicating good sensitivity of the miniature pig kit. The SNP genotyping results for four miniature pig DNA samples at a concentration of 40 μg / mL were completely consistent, demonstrating strong reproducibility of the miniature pig kit (Table 6). Specificity verification of the miniature pig kit using 40 μg / mL miniature pig DNA revealed that the kit could not amplify or genotype SNP sites in Chinese hamster DNA samples, indicating good specificity of the miniature pig kit. In conclusion, the miniature pig kit demonstrates good performance in terms of sensitivity, specificity, and reproducibility.

[0111] Table 6. Validation results of the miniature pig test kit

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[0118] 7. Popgen.32 population genetic structure analysis

[0119] To further optimize the SNP locus combinations, this application performed genotyping on 221 SNP loci from five miniature pig populations. The results showed that the MAPQ (mapping quality) values ​​of three SNP loci (MP160, MP166, and MP218) were less than 59, indicating that the reads at these three loci did not have unique positions on the reference genome, making the genotyping results unreliable. This application also found that one SNP locus, MP131, was monomorphic in all miniature pig samples, with a genotype of T / T. This application concludes that such loci with limited polymorphism are unsuitable for subsequent population analysis.

[0120] In addition, this study also identified several linkage loci (MP36, MP39, MP53, MP54, MP189, MP221, MP222) and loci with poor sequencing quality (MP21 and indel locus MP76), which were subsequently optimized. In summary, this application ultimately selected 207 high-quality SNP loci and recommends them for genetic evaluation in miniature pig populations.

[0121] This application used Popgen.32 software to analyze the genetic structure of four closed miniature pig populations using combinations of 207 SNP loci with high amplification quality and good polymorphism from the kit (Table 7). The results show that the HN and TZ populations have relatively high values ​​for various genetic parameters, including mean effective allele count (ne*), Shannon index (I*), observed heterozygosity (ObsHet*), expected heterozygosity (ExpHet*), mean heterozygosity (AveHet*), and polymorphism information content (PIC). This is followed by the HSY population, and finally the YNF population.

[0122] Table 7. Genetic analysis of four closed groups of miniature pigs.

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[0124] This application constructed genetic tree diagrams for four closed populations of miniature pigs based on Nei genetic distance and UPGMA methods. It was found that the HN population was closest in genetic distance to the TZ population, and they were furthest from the YNF population. Figure 3 ).

[0125] 8. Principal Component Analysis (PCA)

[0126] This study used principal component analysis (PCA) on dimensionality-reduced data to calculate the genetic distance between complex samples. The PCA results based on the allele frequency distribution of 207 polymorphic SNP loci in four miniature pig closed populations are as follows: Figure 4 As shown. In the genotype-based PCA results, the HN and TZ populations were difficult to separate, while the YNF and HSY populations were well separated from other populations. Figure 4 A). The PCA 3D diagram can distinguish the four closed groups of miniature pigs, but the HN and TZ groups are still the closest to each other. Figure 4 B). These are all consistent with the results of the genetic tree diagram.

[0127] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A mini-pig SNP detection kit, characterized in that, The kit is used for detecting a small pig SNP molecular marker combination; The small pig SNP molecular marker combination is composed of 208 SNP molecular markers, physical positions of the 208 SNP molecular markers are determined by sequence alignment based on whole genome sequencing with a coverage of 10x of small pigs, and site information of the 208 SNP molecular markers is shown in Table 1: Table 1 208 SNP site information of small pigs 2. The miniature pig SNP detection kit according to claim 1, characterized in that, The kit contains a primer set and / or a probe for detecting the small pig SNP molecular marker combination.

3. A miniature pig SNP gene detection system, characterized in that, The small pig SNP detection kit according to any one of claims 1-2.

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