Microsatellite loci combination and primers for Przewalski's horse and their application

Abstract

The present invention discloses a combination of microsatellite loci of Przewalski's horse and primers and applications thereof, and belongs to the technical field of DNA molecular markers. The combination of microsatellite loci of Przewalski's horse includes 16 microsatellite loci, and the nucleotide sequences of the 16 microsatellite loci are respectively shown as SEQ ID NO.33‑48. The present invention uses a bioinformatics method to screen the DNA sequence containing microsatellites in the reference genome of Przewalski's horse, designs specific primers, performs polymorphism detection on these microsatellite loci, develops a group of Przewalski's horse microsatellite molecular markers with rich polymorphism, and provides a group of Przewalski's horse microsatellite primers. The Przewalski's horse microsatellite primer pair provided by the present invention can specifically amplify the microsatellite loci, and has a high degree of polymorphism, and can be applied to research fields such as genetic diversity, population genetic structure, phylogenetic geography, evolution and kinship of Przewalski's horse.

Description

Technical Field

[0001] The invention relates to the technical field of DNA molecular markers, in particular to a Przewalski's horse microsatellite locus combination, primers and applications thereof. Background Art

[0002] Microsatellite: Microsatellite (simple sequence repeat, SSR) refers to a simple tandem repeat sequence of DNA, which consists of a core sequence and flanking sequences on both sides. The core sequence is a repeating unit of 1-6 nucleotides. The differences in the number of repeats and repeating units between different alleles make it length polymorphic. The two sides of the core sequence are composed of conservative flanking sequences, which have high homology between species and can therefore be used for primer design. Compared with other molecular markers, microsatellites have the advantages of large number, good stability, rich polymorphism information, co-dominance and easy detection. At the same time, microsatellite analysis based on PCR technology has low requirements for template DNA, and nanogram amounts or partially broken DNA can be effectively analyzed. Therefore, microsatellite markers are increasingly used in genetic research on endangered species, such as explaining the evolutionary history of endangered species, analyzing population genetic structure, assisting population surveys, identifying kinship relationships, and identifying closely related species and hybrid individuals, and other key conservation biology tasks. However, although some studies have found that microsatellite DNA flanking sequences are somewhat conserved among closely related species, for some rare and endangered species, when there is a lack of reference microsatellite sequence information of related closely related species, it is necessary to screen microsatellite loci and develop specific primers. In addition, the genetic diversity of endangered species is generally low, so the development of polymorphic microsatellite loci is more difficult and more important.

[0003] Przewalski's Horse Equusferus Przewalskii belongs to the genus Equidae of the order Perissodactyla. It is a national first-class protected wild animal and the only remaining wild horse population in the world. Due to the development of pastoral civilization and population growth, the habitat of Przewalski's horses has been continuously compressed. At present, the number of Przewalski's horses with pedigree records in the world has exceeded 2,000. They have experienced a bottleneck effect and most of them are in captivity and habitat isolation, with serious inbreeding. Research on captive and wild Przewalski's horses is mostly limited to diet and home range analysis, and there are few studies using molecular marker technology to analyze and evaluate their genetic diversity in detail. Therefore, it is crucial to develop efficient and powerful molecular markers to identify individuals of reintroduced Przewalski's horses and analyze their genetic diversity. Summary of the invention

[0004] The purpose of the present invention is to provide a Przewalski's horse microsatellite locus combination and primers and applications thereof, so as to solve the problems existing in the above-mentioned prior art. The Przewalski's horse microsatellite primer pair provided by the present invention can specifically amplify the microsatellite locus and has high polymorphism, can be applied to research fields such as genetic diversity, population genetic structure, phylogenetic geography, evolution and kinship of Przewalski's horse, has good repeatability, and is a reliable and effective molecular marker.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a Przewalski's horse microsatellite locus combination, comprising 16 microsatellite loci, and the nucleotide sequences thereof are respectively shown as SEQ ID NO.33-48.

[0007] The present invention also provides a primer set for amplifying the above-mentioned Przewalski's horse microsatellite locus combination, and the primer set includes the nucleotide sequences shown as SEQ ID NO.1-32.

[0008] The present invention also provides a Przewalski's horse microsatellite kit, comprising the above primer set.

[0009] The present invention also provides a method for detecting microsatellite loci of Przewalski's horse, comprising the following steps:

[0010] S1, extracting genomic DNA of Przewalski's horse;

[0011] S2. Synthesizing fluorescent primers from the primer set shown in SEQ ID NO. 1-32 to obtain a fluorescently labeled Przewalski's horse microsatellite primer set;

[0012] S3, using the Przewalski's horse genomic DNA in S1 as a template, and using the fluorescently labeled Przewalski's horse microsatellite primer set in S2 to perform PCR amplification to obtain an amplified product;

[0013] S4, performing gel electrophoresis and capillary electrophoresis typing detection on the PCR amplification products in S3;

[0014] S5. Evaluate the polymorphism of microsatellite loci based on the results detected in S4.

[0015] Furthermore, in step S2, the fluorescently labeled Przewalski's horse microsatellite primer set is obtained by labeling the 5' end of a single primer of the primer set shown in SEQ ID NO. 1-32 with FAM.

[0016] Furthermore, in step S1, the Przewalski's horse genomic DNA is DNA extracted from feces.

[0017] Furthermore, in step S3, the PCR amplification program is: 94°C pre-denaturation for 2-5 min, 94°C denaturation for 30 s, 50°C-60°C annealing for 30 s, 72°C extension for 90 s, a total of 30 cycles, and finally 72°C repair extension for 5-10 min.

[0018] Furthermore, in step S3, the PCR amplification system is: 1 μL of template DNA, 1 μL of 10 μM forward and reverse primers, 12.5 μL of 2×Easy TaqR PCR SuperMix, and 9.5 μL of Nuclease-free Water.

[0019] The present invention also provides an application of the Przewalski's horse microsatellite locus combination or the primer set or the Przewalski's horse microsatellite kit in Przewalski's horse population analysis.

[0020] Furthermore, the Przewalski's horse population analysis includes genetic diversity, population genetic structure, phylogenetic geography or parentage identification of Przewalski's horses.

[0021] The present invention discloses the following technical effects:

[0022] The present invention uses a bioinformatics method to screen the DNA sequences containing microsatellites in the reference genome of Przewalski's horse, designs specific primers, performs polymorphism detection on these microsatellite sites, develops a group (16) of Przewalski's horse microsatellite molecular markers with rich polymorphism, and provides a group of Przewalski's horse microsatellite primers. The Przewalski's horse microsatellite primer pair provided by the present invention can specifically amplify the microsatellite site and has high polymorphism, can be applied to the research fields such as genetic diversity, population genetic structure, phylogenetic geography, evolution and kinship of Przewalski's horse, has good repeatability, and is a reliable and effective molecular marker. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is the SSR typing diagram of 16 Przewalski's horse samples DNA amplified by primers at YMYW01 locus;

[0025] Figure 2 This is the SSR typing diagram of 16 Przewalski's horse DNA samples amplified by primers at YMYW12 locus;

[0026] Figure 3This is the SSR typing diagram of 16 Przewalski's horse DNA samples amplified by primers at YMYW13 locus;

[0027] Figure 4 This is the SSR typing diagram of 16 Przewalski's horse samples DNA amplified by primers at YMYW21 locus;

[0028] Figure 5 This is the SSR typing diagram of DNA from 16 Przewalski's horse samples amplified by primers at YMYW22 locus;

[0029] Figure 6 This is the SSR typing diagram of 16 Przewalski's horse DNA samples amplified by primers at YMYW186 locus;

[0030] Figure 7 This is the SSR typing diagram of 16 Przewalski's horse DNA samples amplified by primers at YMYW666 locus;

[0031] Figure 8 This is the SSR typing diagram of 16 Przewalski's horse samples DNA amplified by primer YMYW0104;

[0032] Fig. 9 This is the SSR typing diagram of DNA from 16 Przewalski's horse samples amplified by primers at YMYW0815 locus;

[0033] Fig.10 This is the SSR typing diagram of 16 Przewalski's horse samples DNA amplified by primers at YMYW0818 locus;

[0034] Fig.11 This is the SSR typing diagram of 16 Przewalski's horse DNA samples amplified by primers at YMYW0829 locus;

[0035] Fig.12 This is the SSR typing diagram of 16 Przewalski's horse DNA samples amplified by primers at YMYW1008 locus;

[0036] Fig.13 This is the SSR typing diagram of 16 Przewalski's horse DNA samples amplified by primers at YMYW1101 locus;

[0037] Fig.14 This is the SSR typing diagram of 16 Przewalski's horse DNA samples amplified by primers at YMYW1201 locus;

[0038] Fig.15 This is the SSR typing diagram of 16 Przewalski's horse DNA samples amplified by primers at YMYW1213 locus;

[0039] Fig.16 This is the SSR typing diagram of the DNA of 16 Przewalski's horse samples amplified by primers at YMYW12138 locus. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] Example 1 Preparation of polymorphic microsatellite markers for Przewalski's horses and primer design

[0045] 1. Extraction of microsatellite loci and primer design

[0046] Based on the genome of Przewalskii (Equus ferus przewalskii) (GCF_000696695.1, https: / / www.ncbi.nlm.nih.gov / genome / ?term=Equus+ferus+przewalskii), SSRsearch software was used to detect microsatellite loci in contigs, and simple repeat sequences that were too close were filtered out. Microsatellite sequences on the genome of Przewalskii were screened. The motifs of microsatellite sequences with repeat units of 2-6 bp must meet the following requirements: for di-base motifs, the number of repetitions must be greater than or equal to 4 times; for tri-base, tetra-base, penta-base and hexa-base motifs, the number of repetitions of the repeat unit must be greater than or equal to 4 times; the minimum length of the SSR sequence is 12 bp; the length of the upstream and downstream sequences of the SSR is 100 bp; the minimum distance between two SSRs is 12 bp. Thus, the microsatellite sequences on the Przewalski's horse genome were identified, and the flanking sequences of 100 bp on both sides of the microsatellite core sequence were extracted for primer design. The number of SSRs finally obtained was 296,131, of which 285,852 had primer fragments. The fragment primer design rate was 96.53%. Primer 3.0 software was used to design primers on both sides of the SSR site, and 285,852 pairs of primer sequences were finally obtained.

[0047] 2. Screening of polymorphic microsatellite primers

[0048] 1) Extraction of fecal DNA: The genomic DNA in feces was extracted using the QIAamp Fast DNA Stool Mini Kit (Germany). A total of 16 DNA samples were extracted, and the collected DNA solution was divided into two equal parts, labeled with number information, and the mother solution was stored at -80°C for future molecular experiments, and the sub-liquid was stored at -20°C for subsequent molecular experiments to construct a DNA template library for Przewalski's horse.

[0049] 2) 20 pairs of SSR primers were randomly selected from the designed microsatellite primers for synthesis, and the 5' end of the single primer was fluorescently labeled (FAM-blue). Templates with better quality were selected from the Przewalski's horse DNA template library (containing 16 fecal DNA samples of Przewalski's horses, and DNA quality was detected by agarose electrophoresis) for primer amplification specificity detection and initial polymorphism screening.

[0050] PCR amplification used the 2×EasyTaq PCR SuperMix kit produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. The amplification system and reaction procedure were as follows:

[0051] 25μL reaction system: 1μL template DNA, 1μL each of 10μM forward and reverse primers, 12.5 μL 2×EasyTaqRPCR SuperMix, 9.5μL Nuclease-free Water.

[0052] The PCR amplification program was as follows: pre-denaturation at 94°C for 2-5 min, denaturation at 94°C for 30 s, annealing at 50°C-60°C for 30 s, extension at 72°C for 90 s, for a total of 30 cycles, and finally repair extension at 72°C for 5-10 min.

[0053] 3) The PCR reaction products were subjected to 2% agarose gel electrophoresis at 120 V for 30 min. After the electrophoresis, the agarose gel was exposed for detection, and clear bands were screened out based on the exposure results. The optimal annealing temperature for each pair of primers was determined based on the clarity of the bands.

[0054] 4) After PCR amplification, the target product was sent to the company (Shanghai Shenggong Bioengineering Co., Ltd.) for capillary electrophoresis typing of microsatellite marker alleles. The electrophoresis raw data results (.FSA) were manually verified and confirmed by GeneMarker v2.2.0 (SoftGenetics LLC., USA), and the SSR amplification bands were finally interpreted. 16 stable microsatellite loci were obtained, marked as YMYW01, YMYW12, YMYW13, YMYW21, YMYW22, YMYW186, YMYW666, YMYW0104, YMYW0815, YMYW0818, YMYW0829, YMYW1008, YMYW1101, YMYW1201, YMYW1213, YMYW12138, and their nucleotide sequences are shown in SEQ ID NO.33-48 respectively; the 16 microsatellite loci and the corresponding primer sequences are shown in Table 1:

[0055] Table 1 16 microsatellite loci and corresponding primer sequences

[0056]

[0057]

[0058]

[0059] Table 2 Nucleotide sequences of 16 microsatellite loci

[0060]

[0061]

[0062] Example 2 Identification of polymorphism of microsatellite markers in Przewalski's horses

[0063] 1. Synthesize fluorescent primers for the 16 pairs of primers corresponding to the 16 microsatellite loci screened in Example 1, i.e., perform fluorescent labeling (FAM-blue) on the 5' end of the single primer; perform PCR amplification using the 16 fecal DNAs of Przewalski's horses in the Przewalski's horse DNA template library constructed in Example 1 as templates, and the amplification system and reaction conditions are the same as those in Example 1. The amplified products are detected by capillary fluorescence electrophoresis on an ABI 3730XL sequencer, and the allele and other data are counted based on the electrophoresis results. The SSR typing diagram of 16 Przewalski's horse samples amplified by 16 pairs of primers corresponding to 16 microsatellite loci is shown in Figure 1-Figure 16 , S1~S16 in the figure represent 16 Przewalski's horse samples respectively.

[0064] GeneMaker software was used to separate the collected data by fluorescence color, calculate the allele size, and then determine the genotyping data of each locus. The GenAlex plug-in and Cervus were used to determine the genetic index to further evaluate the polymorphism of the microsatellite loci. The polymorphism detection results (Table 3) are shown.

[0065] Table 3 Polymorphism of 16 microsatellite loci in Przewalski's horse

[0066]

[0067] Note: K represents the number of alleles; N represents the number of genotyped individuals; Ho represents the observed heterozygosity; He represents the expected heterozygosity; PIC represents the polymorphic information content.

[0068] Table 3 shows that the present invention uses 16 pairs of primers corresponding to 16 microsatellite loci to amplify 16 Przewalski's horse samples, and the results of genetic diversity analysis show that the number of alleles (K) at each locus is 1 to 4, the observed heterozygosity (Ho) is 0.0 to 0.8, the expected heterozygosity (He) is 0.0 to 0.8, and the polymorphic information content (PIC) is 0.0 to 0.7. It can be seen that the 16 microsatellite molecular markers of the present invention have good polymorphism and high specificity, and can be used for genetic diversity, population genetic structure, phylogenetic geography, evolution and kinship research, etc., and have good application prospects.

[0069] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention. Sequence Listing <110> Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences Xinjiang Uygur Autonomous Region Wild Horse Breeding Research Center <120> Microsatellite loci combination and primers for Przewalski's horse and their application <160> 48 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty two <212> DNA <213> Artificial Sequence <400> 1 gcctgtacaa gtgggttagt at 22 <210> 2 <211> twenty three <212> DNA <213> Artificial Sequence <400> 2 ccatttcctg ttttattttt gta 23 <210> 3 <211> twenty three <212> DNA <213> Artificial Sequence <400> 3 ggaatagaat ttagggaaga aga 23 <210> 4 <211> twenty three <212> DNA <213> Artificial Sequence <400> 4 aagtcagtca acatccaata aac 23 <210> 5 <211> twenty three <212> DNA <213> Artificial Sequence <400> 5 catacaacag aaatttcaca atg 23 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <400> 6 gaataacctc atttcacagg taa 23 <210> 7 <211> twenty three <212> DNA <213> Artificial Sequence <400> 7 gagcataatc cttgcttaca tac 23 <210> 8 <211> twenty three <212> DNA <213> Artificial Sequence <400> 8 acttagcaag tacctgttgg ata 23 <210> 9 <211> twenty three <212> DNA <213> Artificial Sequence <400> 9 gactgaattt ttgttctgtt ttc 23 <210> 10 <211> twenty three <212> DNA <213> Artificial Sequence <400> 10 tgtgcctaga aagtagtagg tgt 23 <210> 11 <211> twenty three <212> DNA <213> Artificial Sequence <400> 11 acaagactga ggtctttaag tca 23 <210> 12 <211> twenty three <212> DNA <213> Artificial Sequence <400> 12 gtatgagaaa ggaaagagga aat 23 <210> 13 <211> twenty three <212> DNA <213> Artificial Sequence <400> 13 cctggtctct ataccaatat caa 23 <210> 14 <211> twenty two <212> DNA <213> Artificial Sequence <400> 14 gtttaggtaa gggaggagtt tt 22 <210> 15 <211> twenty three <212> DNA <213> Artificial Sequence <400> 15 ctggatcata tggtgtttct att 23 <210> 16 <211> twenty three <212> DNA <213> Artificial Sequence <400> 16 cagaaggacc tacaactaga atg 23 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <400> 17 gttgggagaa gactaccagg 20 <210> 18 <211> twenty two <212> DNA <213> Artificial Sequence <400> 18 ctccagagtt caagtagaca gc 22 <210> 19 <211> twenty three <212> DNA <213> Artificial Sequence <400> 19 ccattttgta aatacacatg agc 23 <210> 20 <211> twenty three <212> DNA <213> Artificial Sequence <400> 20 gaagaagtaa ttcaaagtca ccc 23 <210> twenty one <211> twenty two <212> DNA <213> Artificial Sequence <400> twenty one ctgagaatct gcatttctaa cc 22 <210> twenty two <211> twenty three <212> DNA <213> Artificial Sequence <400> twenty two agtattcttc ttaagtgcat ccc 23 <210> twenty three <211> twenty three <212> DNA <213> Artificial Sequence <400> twenty three ctacatcaga accaaccata aag 23 <210> twenty four <211> twenty two <212> DNA <213> Artificial Sequence <400> twenty four atgatcaaaa ctggttaagg tg 22 <210> 25 <211> twenty three <212> DNA <213> Artificial Sequence <400> 25 gagaggaaat cactttctga tct 23 <210> 26 <211> twenty three <212> DNA <213> Artificial Sequence <400> 26 taggaagaac acttccactt aca 23 <210> 27 <211> twenty two <212> DNA <213> Artificial Sequence <400> 27 gaattaatta cagcaaggga tg 22 <210> 28 <211> twenty two <212> DNA <213> Artificial Sequence <400> 28 ggtggaaacg actctaatga ta 22 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <400> 29 acatttgttt gggggttttt <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <400> 30 acacataca aagggagga gat <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <400> 31 ctagtcccct catcccagag <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <400> 32 acaggtgtta tcgaggacaa ag <210> 33 <211> 214 <212> DNA <213> Artificial Sequence <400> 33 agtatgaact catatttagc ttaattcata ttcagatgga taaatacaga aacaatgata aataggcctg tacaagtggg ttagtatata tgtatctgag father father fathercata tacctaagtg tgcatatatg tatgtgtgtg tgtatatata father's acaaaaataa aacaggaaat ggatcataga cataaatgta agag <210> 34 <211> 212 <212> DNA <213> Artificial Sequence <400> 34 aagctggggc tgagatcgct tctttcatgt ctggaataga atttagggaa gaagacttag 60 gtcttggctt ttctttggga ccatcctgtc agtgtttcag attattatta tttgggaaaa 120 aaaaatagtc ctgaggcctt aagaagccag agaacagttg tttattggat gttgactgac 180 ttaaacatgt gaagacatag ggtgcaaagg ag 212 <210> 35 <211> 216 <212> DNA <213> Artificial Sequence <400> 35 tggggttggt acaggggtga aggggagcac ttatatggtg acagacaaga aataacatac 60 aacagaaatt tcacaatgat gtaaactatt ataaacacaa taattaatta attaattagc 120 ccatgatttt acctgtgaaa tgaggttatt ctggaatagc cagagaattg caattcggga 180 tatgcatact atggcagact ataggcaaat ccaaca 216 <210> 36 <211> 216 <212> DNA <213> Artificial Sequence <400> 36 aaaaagccct tttccaagaa gaataagtaa agcagataaa ctggaacatg tttcaggtta 60 gagcataatc cttgcttaca tacatctgtc cattcatcag tccatccatc catccatcta 120 tccatctatc caacaggtac ttgctaagta tcaggtgcca tgttatagct attttactgt 180 tggctggaaa ggtaggttgg aaccagattt tgaaaa 216 <210> 37 <211> 212 <212> DNA <213> Artificial Sequence <400> 37 tattagttgg gtgtccagca cttttccagg actgaatttt tgttctgttt tctacccctc 60 cccacctaaa ataggtttca tcttcttcct tccttctttc ctctctctct ctttcttttt 120 tccactattt ctcgtgatac acctactact ttctaggcac aagaattagt gaatcatagt 180 ctctgatctg agatttccag ttttatggag aa 212 <210> 38 <211> 216 <212> DNA <213> Artificial Sequence <400> 38 actataaata tacattataa tctacaatgg ctgaatttgt cagagtgaat atgaacttta 60 tggagtctaa gtgagacaat actgaggtct ttaagtcatt tctctctctc tctctctatg 120 tctatcttca gcttactccc atgcttgtcc caggatgagc agaaaaagta atttcctctt 180 tcctttctca tacatcatga agggctgact aaattg 216 <210> 39 <211> 214 <212> DNA <213> Artificial Sequence <400> 39 aacttttggt accactagct aaaactgttt ctccaaggac tacagtgtgc tactctgtcc 60 tccatgtatt ttcagtacct ggtctctata ccaatatcaa tctctctctc tctccacgta 120 ctcctgctat cgttcccagc aacttcaatg ttcctccacc acatcatctt ggcaaaactc 180 ctcccttacc taaaccacac ttcatgacca cccc 214 <210> 40 <211> 212 <212> DNA <213> Artificial Sequence <400> 40 cagtagtagg atagctggat catatggtgt ttctattttt aatttttttt taaagtttgg 60 cacctgagct aacatccatt gccaatcttt tcttcttctt cttcttcttc ttctccctaa 120 agccccccag tacatagctg tacattctag ttgtaggtcc ttctggtagc ctcagcatgg cttgatgagc ggtgccatgt ctgcacccag ga <210> 41 <211> 212 <212> DNA <213> Artificial Sequence <400> 41 actgaggcca accctacctt tggagaggca gggcggggtt gggagagac taccaggccg tccccatgtc caggcgcaga ggtctgcctc ccagcctcag gtgtgtgtgt gttgggggtc 120 ggggagctgt ctacttgaac tctggagagt taaacacagc acgtgggctc cgtggaagga gtttctgttg acaagctggg ctttctcctg ac 212 <210> 42 <211> 224 <212> DNA <213> Artificial Sequence <400> 42 gggaatcatg to be able to tttccatttt gtaaatacac to be able to beat father aaatcattca fatheratttgt atgttcacat attack attack attack attack caaaaaatgc tattcttaat attack gggtgacttt gaattacttc ttctttgtac tttttgcat ttttccaaat tttcttctat aatg <210> 43 <211> 218 <212> DNA <213> Artificial Sequence <400> 43 caaaggaaac atggcaaggt gagtgccaac agcactgctg aagcagattc cgacccagta 60 gggctgggca ggcctgagaa tctgcatttc taaccccact accaccacca ccaccaccag 120 ggatgctcat actcctggtc tgggatgcac ttaagaagaa tactgactgc aaccaggtca 180 cactagcgag tcttctccag atagcacttc tgtgttcg 218 <210> 44 <211> 212 <212> DNA <213> Artificial Sequence <400> 44 attcaagaag ttccttaact ttctagaaga aagcttttca aagcatggac cacggacaac 60 ctacatcaga accaaccata aagcttatta aaaacaagag caacaacaac aacagatctg 120 tcaaaccaga atctctgagg gatggaaccc atgcagctgc accttaacca gttttgatca 180 tgctcagatt tgagaactgt tggtgtcgta ta 212 <210> 45 <211> 224 <212> DNA <213> Artificial Sequence <400> 45 atacagcccc ttggggtcta agtgtaggca gggtaggaag cagtggggca gcctctgatg 60 gtaaccccat cagagaggaa atcactttct gatctgctcg cacacacaca cacacacaca 120 cacacgcatc caccttttgt tctctgtgct gtaagtggaa gtgttcttcc tactttatac 180 atctagatac caagtcagga agtgtgcttt ggatggctga atgc 224 <210> 46 <211> 215 <212> DNA <213> Artificial Sequence <400> 46 catgggacac agctgccctg gtccagggtc agagaagcga attaattaca gcaagggatg 60 cgctagggca ttagtggcga cagacccccc tgagcacagc catcatcatc atcatcgaca 120 cgattatcat tagagtcgtt tccaccacgt ttcttaaaca gcacccgggg gtcccacgag 180 tggccagcca gccttcaatt caggaagcat gtcat 215 <210> 47 <211> 221 <212> DNA <213> Artificial Sequence <400> 47 gactaagtaa ttatccttct ttttatttta taaatcatag attttgttta ttaaaacaac 60 atttgtttgg gggttttttt gtataagtgg ttttgaaaaa attattatta ttattattat 120 tttttcactg aggaagatta gccctgagct aacgtctgca ccgatcttcc tcccttttgt 180 atgtgttttg ctgccacagc atggctgaga agtggtgtag g 221 <210> 48 <211> 214 <212> DNA <213> Artificial Sequence <400> 48 cctagtcccc tcatcccaga gccactcagg ccagatgcga ggctgccgtg ctgtcaaggt 60 gggcagtcag cctcctccct gacacatgca ggtgcacgcg cacacacaca cacacgctca 120 cgcgctttgt cctcgataac acctgttgag gccccacctg acacaggtag gcagagcccc 180 agggacagga tgctgatgtt aagttagcgg tccc 214

Claims

1. A primer set for amplifying a combination of microsatellite loci of Przewalski's horse, characterized in that: The Przewalski's horse microsatellite locus combination includes 16 microsatellite loci, and the nucleotide sequences thereof are respectively shown as SEQ ID NOs.33-48; the primer set includes the nucleotide sequences shown as SEQ ID NOs.1-32.

2. A microsatellite kit for Przewalski's horse, characterized in that: Comprising the primer set according to claim 1.

3. A method for detecting microsatellite loci of Przewalski's horse, characterized in that: The following steps are involved: S1, extracting genomic DNA of Przewalski's horse; S2. Synthesizing fluorescent primers from the primer set shown in SEQ ID NO. 1-32 to obtain a fluorescently labeled Przewalski's horse microsatellite primer set; S3, using the Przewalski's horse genomic DNA in S1 as a template, and using the fluorescently labeled Przewalski's horse microsatellite primer set in S2 to perform PCR amplification to obtain an amplified product; S4, performing gel electrophoresis and capillary electrophoresis typing detection on the PCR amplification products in S3; S5. Evaluate the polymorphism of microsatellite loci based on the results detected in S4.

4. The detection method according to claim 3, characterized in that: In step S2, the fluorescently labeled Przewalski's horse microsatellite primer set is obtained by labeling the 5' end of a single primer of the primer set shown in SEQ ID NO. 1-32 with FAM.

5. The detection method according to claim 3, characterized in that: In step S1, the Przewalski's horse genomic DNA is DNA extracted from feces.

6. The detection method according to claim 3, characterized in that: In step S3, the PCR amplification program is: 94°C pre-denaturation for 2-5 min, 94°C denaturation for 30 s, 50°C-60°C annealing for 30 s, 72°C extension for 90 s, a total of 30 cycles, and finally 72°C repair extension for 5-10 min.

7. The detection method according to claim 3, characterized in that: In step S3, the PCR amplification system is: 1 μL of template DNA, 1 μL of 10 μM forward and reverse primers, 12.5 μL of 2×Easy TaqR PCR SuperMix, and 9.5 μL of Nuclease-free Water.

8. Use of the primer set according to claim 1 or the Przewalski's horse microsatellite kit according to claim 2 in Przewalski's horse population analysis.

9. The use according to claim 8, characterized in that: The Przewalski's horse population analysis includes Przewalski's horse genetic diversity, population genetic structure or phylogeography.

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