Mongolian gerbil 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 meriones unguiculatus SNP molecular marker combination and application thereof. The meriones unguiculatus SNP molecular marker combination disclosed by the invention comprises at least one of 219 SNP molecular markers, the physical positions of the 219 SNP molecular markers are determined by performing sequence alignment on whole genome sequencing with the coverage rate of 10 * on the basis of meriones unguiculatus, and the site information of the 219 SNP molecular markers is shown in a table 1. The meriones unguiculatus SNP molecular marker combination provided by the invention is used for detecting the SNP genotyping of the meriones unguiculatus, 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 in particular to a long-clawed gerbil SNP molecular marker combination and application thereof. Background Art

[0002] Meriones unguiculatus, also known as Mongolian gerbil, belongs to the genus Meriones of the family Cricetidae. It looks a bit like a squirrel, with brown-gray back hair and gray-white belly hair. It is of moderate size, between rats and mice. Wild gerbils are mainly distributed in semi-arid areas such as Liaoning in Northeast my country and Ningxia in Inner Mongolia in Northwest China. The experimental gerbil populations all originated from China. They were captured, domesticated, and bred by Dalian Medical University, Capital Medical University, and Zhejiang Provincial Experimental Animal Center, and research work such as experimental animalization and population cultivation was carried out. In 1935 and 1954, they were introduced to Japan and the United States for domestication, and are currently distributed all over the world. The long-clawed gerbil is a special experimental animal in my country and has a very important value in medical and biological research. Studies have been conducted on Willis circle (CoW) mutations and cerebral ischemia and Helicobacter pylori-related gastric diseases. A gerbil stroke model and a heritable spontaneous diabetes model have also been established. The long-clawed gerbil is sensitive to a variety of microorganisms (Brucella, Hantavirus, etc.) and parasites such as filarial worms and roundworms. The long-clawed gerbil is considered to be the most suitable small animal model for studying listeriosis, because the invasion pathway of Listeria in the long-clawed gerbil is the same as that in the human body. The long-clawed gerbil is the only rodent found to be able to cause gastritis and gastric cancer by long-term infection with Helicobacter pylori (HP). Its immunopathological changes after infection with HP include hypochlorhydria and changes in intestinal flora, which are very similar to those of humans. The long-clawed gerbil has many unique behavioral characteristics. For example, the monogamous long-clawed gerbil shows a high level of paternal behavior, which is an ideal model for studying the regulation of paternal behavior by hormones. The repeated separation of newborn long-clawed gerbils from their mothers and siblings has an impact on their behavior, which will produce some characteristics similar to human depression. In addition, the long-clawed gerbil is an ideal model for studying drugs related to the antidepressant NK (Neurokinin) receptor and SP (Substance P) pathways. Compared to rats, gerbils exhibit more anxiety-like behaviors, more active exploratory behaviors and sensitivity to pain, and also have more social behaviors, which makes gerbils more sensitive to these tests.

[0003] Genetic monitoring is one of the methods to detect the genetic diversity and consistency of genetic background of experimental animals, and it can detect potential genetic mutations and genetic contamination. Unfortunately, the genetic quality detection methods of long-clawed gerbils are very scarce both at home and abroad. At present, the genetic monitoring of long-clawed gerbils in China mainly focuses on biochemical gene loci detection and microsatellite markers, and there are no genetic quality detection methods suitable for long-clawed gerbils internationally. However, the biochemical marker analysis method has a small coverage, low detection rate, and difficulty in judging the results, and has long been eliminated by developed countries; the microsatellite method has a high mutation rate and false detection rate, and cannot achieve high throughput, and the detection efficiency is low.

[0004] Single nucleotide polymorphism (SNP) mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide (A, T, C and G) at the genomic level. As a third-generation genetic marker, single nucleotide polymorphism detection technology has the advantages of accuracy, time saving and suitability for automation, and is being widely used in genetic evaluation of experimental animals. The existing GB14923-2022 "Genetic Quality Control of Laboratory Animals" standard was revised in 2022. The standard recommends 35 SNP marker genes suitable for inbred mice, but does not clearly specify the SNP genetic quality detection method and standard detection system applicable to long-clawed gerbils.

[0005] Therefore, it is urgent to build an internationally advanced SNP genetic quality detection method and standard technical system suitable for long-clawed gerbils. This can not only fill the gaps in relevant standards at home and abroad, but also has important significance for improving the level of genetic detection technology of long-clawed gerbil populations and ensuring the quality of long-clawed gerbil populations. Summary of the invention

[0006] In order to solve the above technical problems, the present application provides a long-clawed gerbil SNP molecular marker combination and its application.

[0007] In the first aspect, the present application provides a combination of SNP molecular markers for gerbils, including at least one of 219 SNP molecular markers, the physical positions of the 219 SNP molecular markers are determined by sequence alignment based on whole genome sequencing of gerbils with a coverage of 10×, and the site information of the 219 SNP molecular markers is shown in Table 1.

[0008] In view of the shortcomings of existing long-clawed gerbil genetic quality detection methods, such as complex operation, time-consuming and labor-intensive, difficult result analysis, and not suitable for high-throughput detection, the purpose of this application is to establish a SNP site genetic detection method suitable for long-clawed gerbil germplasm resources. The long-clawed gerbil SNP molecular marker combination system provided in this application is novel, stable, economical and efficient for SNP site detection in long-clawed gerbils.

[0009] In a second aspect, the present application provides a method for screening the loci of the long-clawed gerbil SNP molecular marker combination, comprising the following steps: (1) Whole-genome sequencing and sequencing quality control of long-clawed gerbils: Whole-genome sequencing with a coverage of 10× was performed on long-clawed gerbils; raw reads were filtered using fastp software; paired end sequences were mapped to the long-clawed gerbil reference genome MunDraft-v1.0 using BWA software; SNPs were called and filtered using GATK4.1 software; data quality control was performed using BCFtools software, and the following standards were set: SNP loci with QualByDepth < 2.0, SNP loci with FisherStrand < 60.0, SNP loci with RMSMappingQuality < 30.0, and SNP loci with sequencing depth < 8.0 were removed; (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.

[0010] In a third aspect, the present application provides a primer set for detecting the gerbil SNP molecular marker combination, and the position information of the primer set is shown in Table 2.

[0011] In a fourth aspect, the present application provides a gerbil SNP detection kit, which is used to detect the gerbil SNP molecular marker combination.

[0012] Preferably, the kit comprises a primer set and / or a probe for detecting the gerbil SNP molecular marker combination.

[0013] In a fifth aspect, the present application provides a gerbil SNP gene detection system, including the gerbil SNP molecular marker combination or the primer set of the gerbil SNP molecular marker combination or the gerbil SNP detection kit.

[0014] Preferably, the long-clawed gerbil SNP gene detection system comprises the following steps in sequence: collecting long-clawed gerbil samples; extracting and authenticating long-clawed gerbil DNA; resequencing the whole genome of long-clawed gerbils; 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 long-clawed gerbil multi-target region capture kit to perform genotyping on all SNP sites; validating the multi-target region capture kit: sensitivity, repeatability, and specificity; and analyzing population genetic structure.

[0015] In a sixth aspect, the present application provides the application of at least one of the long-clawed gerbil SNP molecular marker combination, the primer set, the long-clawed gerbil SNP detection kit, and the long-clawed gerbil SNP gene detection system in the SNP genetic quality detection method and standard technical system of long-clawed gerbils. 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. Based on the results of second-generation sequencing, 219 SNPs sites were preliminarily screened. By genotyping three closed groups, a multiplex PCR amplification system including 206 SNP sites was established and optimized. A repeatable screening standard was established to preliminarily screen out SNPs with a wide distribution and high polymorphism level in the genome. The detection system of this application has the characteristics of novel, stable, cost-effective and efficient for SNP genotyping of long-clawed gerbils. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the specific experimental process of the gerbil SNP gene detection system in this application.

[0017] Figure 2 This is the screening process for SNP molecular markers of gerbils in this application.

[0018] Figure 3 This is the genetic tree diagram of the three closed groups of long-clawed gerbils in this application.

[0019] Figure 4 The two-dimensional (A) and three-dimensional (B) principal component analysis diagrams of the three groups of 206 SNPs in this application. DETAILED DESCRIPTION

[0020] 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

[0021] This embodiment provides a SNP gene detection system for gerbils, and the experimental process is as follows: Figure 1 As shown, the specific steps are described as follows.

[0022] 1. Preparation of Genomic DNA of Gerbils 1.1 Collection of animal samples Three commonly used closed groups of gerbils in China were selected, namely the closed group of gerbils in Dalian Medical University (Dalian, 32), the closed group of gerbils in Zhejiang Animal Experiment Center (Zhejiang, 37) and the closed group of gerbils in Capital Medical University (CMU, 31), as well as two inbred strains of gerbils in Capital Medical University, namely the cerebral ischemia inbred strain (cerebral ischemia, 10) and the diabetic inbred strain (diabetes, 10). This experiment has been approved by the Animal Experiment and Experimental Animal Welfare Committee of Capital Medical University (AEEI-2021-309).

[0023] 1.2 Preparation of gerbil genomic DNA DNA extraction kit ( DNA was extracted from gerbil ear and tail tissues using the Cell / Tissue DNA Isolation Mini Kit.

[0024] (1) Take 1-20 mg of chopped or ground tissue (liver, spleen and kidney less than 10 mg) and place it in a 1.5 ml centrifuge tube. Add 230 μl Buffer GA and 20 μl Proteinase K and vortex to mix.

[0025] (2) Place the tissue in a 55°C water bath until the tissue is completely digested. (During digestion, the tissue can be inverted to promote the digestion process. Cut the tissue blocks as finely as possible to shorten the digestion time. Liquid nitrogen grinding, mechanical homogenization, glass homogenization and other methods can be used to shorten the digestion time. The digestion time depends on the type of sample and the homogenization effect. Generally, tissue samples require 0.5-3 hours, and mouse tails require 6-8 hours or overnight digestion).

[0026] (3) If the digestion solution is turbid or contains obvious particles, centrifuge at 12,000 rpm (13,400 × g) for 3 min and transfer the supernatant to a new 1.5 ml centrifuge tube.

[0027] (4) Add 250 μl of Buffer GB to the digestion solution, vortex mix at maximum speed for 20 sec, and incubate in a 70°C water bath for 10 min.

[0028] (5) Add 180 μl of anhydrous ethanol to the digestion solution and vortex mix for 15-20 seconds.

[0029] (6) Place the gDNA Columns adsorption column in the Collection Tubes 2ml collection tube. Transfer the mixture obtained in the previous step (including the precipitate) to the adsorption column. Centrifuge at 12,000rpm (13,400×g) for 1min. If the column is clogged after centrifugation, centrifuge at the highest speed for 3-5min. If the mixture exceeds 750μl, pass it through the column several times.

[0030] (7) Discard the filtrate and place the adsorption column in a collection tube. Add 500 μl of Washing Buffer A to the adsorption column. Centrifuge at 12,000 rpm (13,400 × g) for 1 min.

[0031] (8) Discard the filtrate and place the adsorption column in a collection tube. Add 650 μl of Washing Buffer B to the adsorption column. Centrifuge at 12,000 rpm (13,400 × g) for 1 min.

[0032] (9) Repeat step 4.

[0033] (10) Discard the filtrate and place the adsorption column in a collection tube. Centrifuge the empty tube at 12,000 rpm (13,400 × g) for 2 min.

[0034] (11) Place the adsorption column in a new 1.5 ml centrifuge tube. Add 30-100 μl of Elution Buffer preheated to 70°C to the center of the membrane of the adsorption column and place at room temperature for 3 minutes. Centrifuge at 12,000 rpm (13,400 × g) for 1 minute.

[0035] (12) The collected DNA samples were detected using a Nanodrop 2000C micro-volume spectrophotometer.

[0036] (13) Select DNA samples with good absorbance, i.e., A260 / A280 ratio of 1.8 to 2.0. After agarose gel electrophoresis is qualified, store the DNA at 2 to 8°C. For long-term storage, place it at -30°C to -15°C.

[0037] 2. Methods for whole genome sequencing and SNP locus screening of gerbils 2.1 Whole-genome sequencing of long-clawed gerbils and sequencing quality control Due to the limited information in the long-clawed gerbil SNP database, this application performed whole-genome sequencing with a coverage of 10× on 20 Zhejiang gerbils and 20 Capital Medical University gerbils, aiming to find potential polymorphic SNP sites through sequence alignment.

[0038] (1) Whole genome sequencing was performed at a coverage of 10× on 20 Zhejiang gerbils and 20 Capital Medical University gerbils.

[0039] (2) Fastp software (https: / / github.com / OpenGene / fastp) was used to filter the raw reads.

[0040] (3) Using BWA software (https: / / sourceforge.net / projects / bio-bwa / files / ), the paired end sequences were mapped to the reference genome of the gerbil, MunDraft-v1.0 (https: / / www.ncbi.nlm.nih.gov / genome / gdv?org=meriones-unguiculatus&group=muridae).

[0041] (4) GATK4.1 software (https: / / gatk.broadinstitute.org) was used to call and filter SNPs.

[0042] (5) The data quality was controlled using BCFtools software (http: / / www.htslib.org / download / ) and the following standards were set: (5.1) Remove SNP loci with QualByDepth < 2.0; (5.2) Remove SNP loci with FisherStrand<60.0; (5.3) Remove SNP loci with RMSMappingQuality < 30.0; (5.4) Remove SNP loci with sequencing depth <8.0.

[0043] 2.2SNP 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.

[0044] This study followed this strict screening process to screen out high-quality SNP loci combinations suitable for genetic quality identification of long-clawed gerbils.

[0045] By analyzing the second-generation sequencing data of 40 long-clawed gerbils, 944,066 SNP sites were counted in the non-intronic regions and 2,909,545 SNP sites were counted in the intronic regions. Through an efficient SNP screening process, this study ultimately identified 219 high-quality SNP sites for the development of a long-clawed gerbil SNP detection kit ( Figure 2 , Table 1).

[0046] Table 1 Information on 219 SNP loci in gerbils 3. Multiplex PCR kit primer design and genotyping for all SNP loci 3.1 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.

[0047] (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.

[0048] (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.

[0049] (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.

[0050] (5) The instructions for the Long-clawed Gerbil 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.

[0051] Take out Enhancer buffer M from the 4°C refrigerator and thaw at room temperature.

[0052] Prepare the reaction system according to Table 2 on an ice box: Table 2 First round multiplex PCR reaction system Reactants Volume (μL) 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] Centrifuge briefly and place the PCR tube on a magnetic rack for 3 minutes to wait for the solution to clarify.

[0057] 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.

[0058] Centrifuge briefly and place the PCR tube on the DynaMag-96 Side magnetic stand for 3 minutes.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] Centrifuge briefly and place the PCR tube on a magnetic rack for 2 minutes to wait for the solution to clarify.

[0065] 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.

[0066] 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 Reactants Volume (μL) Multiplex PCR products purified in the previous step 13.5 Enhancer buffer M 2.5 Premixed adapter primers, concentration 10 μM each 1 IGT-EM808polymerase mixture 10 ddH2O 3 total 30 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] Centrifuge briefly and place the PCR tube on a magnetic rack for 3 minutes to wait for the solution to clarify.

[0071] 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.

[0072] Centrifuge briefly and place the PCR tube on the DynaMag-96 Side magnetic stand for 3 minutes.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] Centrifuge briefly and place the PCR tube on a magnetic rack for 2 minutes to wait for the solution to clarify.

[0079] 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.

[0080] 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.

[0081] (6) The quality of the PCR primers for the long-clawed gerbil was verified, the primer dimerization and nonspecific amplification were evaluated, the designed and synthesized primers were tested, and the primers with poor detection effects were replaced. Finally, the matching degree of 219 primers on the genome reached 100%. The length of the amplified fragment ranged from 160 bp to 260 bp (Table 4).

[0082] Table 4 Positions and predicted coverage of PCR primers in gerbils 3.2 Validation of the Multi-target Region Capture Sequencing Kit for Gerbils from Multiple Institutions Three companies (Aijitaikang, Tianyi Huiyuan, and Zhongke Guobang) simultaneously verified the long-clawed gerbil SNP locus genetic detection kit, as shown in Table 5 below, and verified the sensitivity, specificity, and repeatability of the long-clawed gerbil kit: Table 5 Validation of the Gerbil Test Kit The experimental results of the three units showed that when the concentration of gerbil DNA was as low as 10 μg / mL, the kit could still perform multiple PCR amplification on the SNP combination of gerbils and accurately type the SNP sites, which showed that the sensitivity of the gerbil kit was good; the typing results of the SNP sites of the four gerbil DNA samples with a concentration of 40 μg / mL were completely consistent, which showed that the gerbil kit had strong repeatability (Table 6); the experiment used 40 μg / mL miniature pig DNA to verify the specificity of the gerbil kit and found that the gerbil kit could not amplify and type the SNP sites of miniature pig DNA samples, which showed that the specificity of the gerbil kit was good. In summary, the gerbil kit performed well in terms of sensitivity, specificity and repeatability.

[0083] Table 6 Validation of the Gerbil Test Kit 4. Analysis of kit application examples 4.1 Analysis of population genetic structure In order to further optimize the efficient SNP site combination, the present application conducted genotyping tests on 219 SNP sites from 5 long-clawed gerbil 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 sites were not unique when compared to the reference genome, and the typing results were unreliable. The present application also found that 7 SNP loci (MG13, MG34, MG72, MG96, MG131, MG199 and MG207) were monomorphic in all gerbil samples (Table 3.3.2). The present application believes that these loci with limited polymorphism are not suitable for subsequent population analysis.

[0084] In addition, this study also found two groups of linkage loci MG139, MG175, MG181 and MG143, MG146, which were subsequently optimized. In summary, this application finally screened out 206 high-quality SNP loci and recommended them for genetic evaluation of the long-clawed gerbil population.

[0085] The application uses Popgen.32 software, and the genetic structure (table 7) of three closed groups of long-clawed gerbils is analyzed by combining 206 high-quality amplification and good polymorphic SNP sites in the test kit. The result of study of the application shows that the long-clawed gerbils from Capital Medical University have the largest population genetics parameter values, including average effective allele number (ne*), Shannon index (I*), observed heterozygosity (Obs Het*), expected heterozygosity (Exp Het*), average heterozygosity (Ave Het*) and polymorphic information content (PIC). Then there are the closed groups of Zhejiang gerbils and the closed groups of Dalian gerbils.

[0086] Table 7 Genetic analysis of three closed populations of Gerbils This application constructed a genetic tree diagram of three closed populations of long-clawed gerbils based on Nei genetic distance and UPGMA method, and found that the Dalian gerbil population had the greatest genetic difference with other gerbil populations. In contrast, the genetic distance between the Zhejiang gerbil population and the Capital Medical University gerbil population was closer ( Figure 3 ).

[0087] 4.2 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 206 polymorphic SNP loci in three closed populations of gerbils are shown in Figure 2. Figure 3 As shown. In the PCA results based on genotypes, PC1 (21.5%) and PC2 (8.2%) accounted for 29.7% of the overall genetic variation. In the PCA two-dimensional diagram, the PC1 dimension can completely distinguish the Dalian gerbil population ( Figure 4 A). The PCA three-dimensional plot can distinguish the three closed groups of gerbils ( Figure 4 B). In addition, the results of principal component analysis showed that the genetic distance between the Zhejiang gerbil closed population and the Shouyi gerbil closed population was closer.

[0088] 4.3 Specific loci of inbred strains of gerbils Among the 206 SNP sites, 156 sites were monomorphic in the cerebral ischemia inbred line, 77 sites were monomorphic in the diabetic inbred line, and 64 sites were monomorphic in both the cerebral ischemia and diabetic inbred lines. Among them, 15 sites showed different genotypes between the two inbred lines and could be used to distinguish the two inbred lines (Table 8).

[0089] Table 8 Fifteen loci with different phenotypes between two inbred populations 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 combination of SNP molecular markers for gerbils, characterized in that: Including at least one of 219 SNP molecular markers, the physical positions of the 219 SNP molecular markers are determined by sequence alignment based on whole genome sequencing of long-clawed gerbils with a coverage rate of 10×, and the site information of the 219 SNP molecular markers is shown in Table 1: Table 1 Information on 219 SNP loci in gerbils 2. The method for screening the gene loci of the long-clawed gerbil 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 long-clawed gerbils: Whole-genome sequencing with a coverage of 10× was performed on long-clawed gerbils; raw reads were filtered using fastp software; paired end sequences were mapped to the long-clawed gerbil reference genome MunDraft-v1.0 using BWA software; SNPs were called and filtered using GATK4.1 software; data quality control was performed using BCFtools software, and the following standards were set: SNP loci with QualByDepth < 2.0, SNP loci with FisherStrand < 60.0, SNP loci with RMSMappingQuality < 30.0, and SNP loci with sequencing depth < 8.0 were removed; (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 gerbil 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 Positions and predicted coverage of PCR primers in gerbils 4. A gerbil SNP detection kit, characterized in that: The kit is used to detect the gerbil SNP molecular marker combination according to claim 1.

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

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

7. The gerbil SNP gene detection system according to claim 6, characterized in that: The method includes the following steps: collecting samples of long-clawed gerbils; extracting and identifying DNA of long-clawed gerbils; and resequencing the whole genome of long-clawed gerbils; Compare sequencing data, perform bioinformatics analysis, and screen SNP candidate sites located in gene regions; Establish SNP screening criteria to form a set of high-quality SNP loci combinations; Multiplex PCR primer design and quality verification for all SNP loci; A multi-target region capture kit for gerbils was developed to perform genotyping on all SNP loci; the multi-target region capture kit was validated in terms of sensitivity, repeatability, and specificity; and the population genetic structure was analyzed.

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

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