A dust mite microsatellite marker, primers and applications thereof, and a method for obtaining the primers

Abstract

The present invention discloses a dust mite microsatellite marker, wherein the microsatellite marker is selected from at least one of Df01, Df02, Df03, Df04, Df05, Df06, Df07, Df08, Df09, Df10, Df11 and Df12, Df01 to Df12 are DNA sequences of SEQ ID NO.01 to Df12 or their complementary sequences, and the microsatellite repeat motifs are (CTG) 5‑8 、(ATG) 5‑7 、(ATC) 9‑12 、(GTT) 6‑7 、(ATC) 6‑8 , (TG) 6‑9 、(ATG) 5‑9 , (GT) 7‑10 、(ATG) 4‑5 、(CAA) 5‑7 、(TTG) 6‑9 and (CA) 7‑8 The microsatellite marker and the primer thereof provided by the present invention have the characteristics of stable PCR amplification, high polymorphism, etc. The primer can be used to analyze the genetic diversity and genetic structure of dust mites, and has high application value.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology DNA markers, and in particular to a dust mite microsatellite marker, a primer and application thereof, and a method for obtaining the primer. Background Art

[0002] Dust mites belong to the family of the family Acaridae and have a wide range of habitats. They often breed in house dust, air conditioning dust screens, flour, biscuit powder, animal feed, Chinese medicinal materials and waste cotton. They are mainly distributed in southern and central China. Dust mites are commonly found in indoor environments where humans live and work. Their excrement, metabolites, shed skins and dead mites are strong allergens that can cause mite allergic diseases, mite dermatitis and mite diseases in the human body.

[0003] At present, there is no specific drug for mite allergic diseases caused by dust mite allergens. Effectively controlling the number of dust mites in the environment has a positive effect on the prevention and treatment of allergic diseases. Research on the genetics of dust mites can provide data support for the formulation of prevention and control strategies for dust mites. At present, there are few studies on the genetics of dust mites, mainly due to the lack of effective molecular markers.

[0004] Microsatellite molecular markers (also known as simple sequence repeats, SSR) are widely distributed in eukaryotic genomes and are composed of tandem repeats of 2 to 6 nucleotides, among which dinucleotides (CA / GT)n are the most common in animals. Compared with other molecular markers, microsatellite molecular markers have the advantages of large number, high polymorphism, good stability, good repeatability, and genetic codominance, and are one of the molecular markers widely used in recent years.

[0005] Therefore, studying polymorphic microsatellite markers can quickly and accurately study the genetic polymorphism and genetic differentiation of dust mite populations, and provide data support for the formulation of control strategies for dust mites. Summary of the invention

[0006] The purpose of the present invention is to provide a dust mite microsatellite marker, primers and applications thereof, and a method for obtaining the primers, so as to solve the technical problems in the prior art.

[0007] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

[0008] The present invention provides a dust mite microsatellite marker, wherein the microsatellite marker is selected from at least one of Df01, Df02, Df03, Df04, Df05, Df06, Df07, Df08, Df09, Df10, Df11 and Df12; wherein,

[0009] The Df01 is a DNA sequence shown in SEQ ID NO.01 or its complementary sequence, and its microsatellite repeat motif is (CTG) 5-8 ;

[0010] The Df02 is a DNA sequence shown in SEQ ID NO.02 or its complementary sequence, and its microsatellite repeat motif is (ATG) 5-7 ;

[0011] The Df03 is a DNA sequence shown in SEQ ID NO.03 or its complementary sequence, and its microsatellite repeat motif is (ATC) 9-12 ;

[0012] The Df04 is a DNA sequence shown in SEQ ID NO.04 or its complementary sequence, and its microsatellite repeat motif is (GTT) 6-7 ;

[0013] The Df05 is a DNA sequence shown in SEQ ID NO.05 or its complementary sequence, and its microsatellite repeat motif is (ATC) 6-8 ;

[0014] The Df06 is a DNA sequence shown in SEQ ID NO.06 or its complementary sequence, and its microsatellite repeat motif is (TG) 6-9 ;

[0015] The Df07 is a DNA sequence shown in SEQ ID NO.07 or its complementary sequence, and its microsatellite repeat motif is (ATG) 5-9 ;

[0016] The Df08 is a DNA sequence shown in SEQ ID NO.08 or its complementary sequence, and its microsatellite repeat motif is (GT) 7-10 ;

[0017] The Df09 is a DNA sequence shown in SEQ ID NO.09 or its complementary sequence, and its microsatellite repeat motif is (ATG) 4-5 ;

[0018] The Df10 is a DNA sequence shown in SEQ ID NO.10 or its complementary sequence, and its microsatellite repeat motif is (CAA) 5-7 ;

[0019] The Df11 is a DNA sequence shown in SEQ ID NO.11 or its complementary sequence, and its microsatellite repeat motif is (TTG) 6-9 ;

[0020] The Df12 is a DNA sequence shown in SEQ ID NO.12 or its complementary sequence, and its microsatellite repeat motif is (CA) 7-8 .

[0021] The present invention also provides a primer for a microsatellite marker of dust mites, wherein the primer comprises at least one of the following 12 pairs of primers, and the 12 pairs of primers are respectively:

[0022] The upstream primer Df01F is shown in SEQ ID NO.13; the downstream primer Df01R is shown in SEQ ID NO.14;

[0023] The upstream primer Df02F is shown in SEQ ID NO.15; the downstream primer Df02R is shown in SEQ ID NO.16;

[0024] The upstream primer Df03F is shown in SEQ ID NO.17; the downstream primer Df03R is shown in SEQ ID NO.18;

[0025] The upstream primer Df04F is shown in SEQ ID NO.19; the downstream primer Df04R is shown in SEQ ID NO.20;

[0026] The upstream primer Df05F is shown in SEQ ID NO.21; the downstream primer Df05R is shown in SEQ ID NO.22;

[0027] The upstream primer Df06F is shown in SEQ ID NO.23; the downstream primer Df06R is shown in SEQ ID NO.24;

[0028] The upstream primer Df07F is shown in SEQ ID NO.25; the downstream primer Df07R is shown in SEQ ID NO.26;

[0029] The upstream primer Df08F is shown in SEQ ID NO.27; the downstream primer Df08R is shown in SEQ ID NO.28;

[0030] The upstream primer Df09F is shown in SEQ ID NO.29; the downstream primer Df09R is shown in SEQ ID NO.30;

[0031] The upstream primer Df10F is shown in SEQ ID NO.31; the downstream primer Df10R is shown in SEQ ID NO.32;

[0032] The upstream primer Df11F is shown in SEQ ID NO.33; the downstream primer Df11R is shown in SEQ ID NO.34;

[0033] The upstream primer Df12F is shown as SEQ ID NO.35; the downstream primer Df12R is shown as SEQ ID NO.36.

[0034] The present invention also provides at least one application of the above-mentioned microsatellite marker or primer in the detection of population genetic diversity and genetic differentiation of dust mites.

[0035] The present invention also provides a method for obtaining dust mite primers, comprising the following steps:

[0036] Step 100, extracting dust mite DNA and sequencing it after passing the test;

[0037] Step 200: Using the existing NCBI dust mite genome sequence as a reference gene, the sequencing data is compared using the BWA software. After correction by the BestPractices process of GATK, the microsatellite markers are searched using the MISA software to screen out polymorphic microsatellite markers and design primers.

[0038] Step 300: Screening out microsatellite markers with an allele greater than 3 from all obtained microsatellite markers based on high-throughput sequencing data of multiple sample genomes;

[0039] Step 400, using Primer 5 software to design primers for the flanking sequences of the microsatellite markers;

[0040] Step 500: Perform PCR amplification using genomic DNA from different dust mite individuals as templates to obtain primers, and perform capillary electrophoresis detection to experimentally verify microsatellite marker primers with strong specificity, good stability and high polymorphism.

[0041] As a preferred solution of the present invention, the sequencing strategy in step 100 is Illumina PE150, and the total sequencing read length is 300 bp.

[0042] As a preferred solution of the present invention, the specific parameters specified when the software MISA performs microsatellite identification analysis in step 200 are:

[0043] A single nucleotide with a single base repeated more than 12 times, a dibase repeated 6 times or more, a tribase or tetrabase repeated 5 times or more, this type of repeat sequence is a microsatellite sequence.

[0044] As a preferred solution of the present invention, the standard for primer design in step 400 is:

[0045] 1) Primer length is 15-30 bp, and the GC content of primers is between 40% and 60%;

[0046] 2) The length of the PCR amplified product is between 100-300 bp;

[0047] 3) The primer base distribution is random, and no more than 4 single bases should appear consecutively;

[0048] 4) The primer does not contain self-complementary sequence;

[0049] 5) There are no more than 4 complementary or homologous bases between a pair of primers;

[0050] 6) There is no complementarity or low complementarity between the forward and reverse primers.

[0051] As a preferred solution of the present invention, the total volume of the PCR reaction for PCR amplification is 12.5 μl, including 6.25 μl 2×Taq PCR Mastermix, 0.5 μl template, and 0.5 μl upstream and downstream primers, and sterilized deionized water is added to make up to 12.5 μl;

[0052] Mix the PCR products evenly and place them in a PCR instrument. Perform pre-denaturation at 94°C for 5 min, denature at 94°C for 30 s, use a temperature gradient to test the optimal annealing temperature, extend at 72°C for 1 min 30 s, cycle 34 times, and finally extend at 72°C for 10 min.

[0053] After the reaction is completed, the PCR products are screened using 1% agarose gel, and the primers with expected length and single band are temporarily stored at 4 degrees Celsius;

[0054] Wherein, the temperature gradient is 51°C, 54°C, 57°C, and 60°C.

[0055] As a preferred solution of the present invention, the size of the primers obtained in step 500 is 20-25 bp. When the annealing temperature is 60° C., the target fragment can be amplified, and the fragment length is about 166-276 bp.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] 1) The microsatellite markers and primers provided by the present invention have the characteristics of stable PCR amplification and high polymorphism, and the primers can be used to analyze the genetic diversity and genetic structure of dust mites, which has high application value, can make up for the shortcomings of microsatellite marker technology in the study of species such as dust mites, and provide support for the study of its application in genetic diversity and genetic differentiation analysis.

[0058] 2) The present invention compares the dust mite genome sequencing read data to the NCBI dust mite reference genome, and then uses MISA software to search and screen microsatellite sites. Then, 50 polymorphic microsatellite markers are screened out based on the high-throughput sequencing data of multiple sample genomes, and then further screened by capillary electrophoresis experiments, and finally 12 highly polymorphic microsatellite markers and primers with high specificity and strong stability are obtained.

[0059] It significantly improves the efficiency of microsatellite marker development and can overcome the shortcomings of conventional polyacrylamide gel electrophoresis and other methods, such as slow analysis speed, cumbersome operation, large errors in quantitative results, and low degree of automation. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0061] Figure 1 The fluorescence scanning peak diagram of the microsatellite locus fragment amplified by the first pair of primers is shown.

[0062] Figure 2 The fluorescence scanning peak diagram of the microsatellite locus fragment amplified by the second pair of primers is shown.

[0063] Figure 3 The fluorescence scanning peak diagram of the microsatellite locus fragment amplified by the third pair of primers is shown.

[0064] Figure 4 The fluorescence scanning peak diagram of the microsatellite locus fragment amplified by the fourth pair of primers is shown.

[0065] Figure 5 The fluorescence scanning peak diagram of the microsatellite locus fragment amplified by the fifth pair of primers is shown.

[0066] Figure 6 The fluorescence scanning peak diagram of the microsatellite locus fragment amplified by the sixth pair of primers is shown.

[0067] Figure 7 The fluorescence scanning peak diagram of the microsatellite locus fragment amplified by the seventh pair of primers is shown.

[0068] Figure 8 The fluorescence scanning peak diagram of the microsatellite locus fragment amplified by the eighth pair of primers is shown.

[0069] Fig. 9 The fluorescence scanning peak diagram of the microsatellite locus fragment amplified by the ninth pair of primers is shown.

[0070] Fig.10 The fluorescence scanning peak diagram of the microsatellite locus fragment amplified by the tenth pair of primers is shown.

[0071] Fig.11 The fluorescence scanning peak diagram of the microsatellite locus fragment amplified by the eleventh pair of primers is shown.

[0072] Fig.12 Shown is the fluorescence scanning peak graph of the microsatellite locus fragment amplified by the twelfth pair of primers. DETAILED DESCRIPTION

[0073] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0074] The present invention provides a dust mite microsatellite marker, wherein the microsatellite marker is selected from at least one of Df01, Df02, Df03, Df04, Df05, Df06, Df07, Df08, Df09, Df10, Df11 and Df12; wherein,

[0075] The Df01 is a DNA sequence shown in SEQ ID NO.01 or its complementary sequence, and its microsatellite repeat motif is (CTG) 5-8 ;

[0076] The Df02 is a DNA sequence shown in SEQ ID NO.02 or its complementary sequence, and its microsatellite repeat motif is (ATG) 5-7 ;

[0077] The Df03 is a DNA sequence shown in SEQ ID NO.03 or its complementary sequence, and its microsatellite repeat motif is (ATC) 9-12 ;

[0078] The Df04 is a DNA sequence shown in SEQ ID NO.04 or its complementary sequence, and its microsatellite repeat motif is (GTT) 6-7 ;

[0079] The Df05 is a DNA sequence shown in SEQ ID NO.05 or its complementary sequence, and its microsatellite repeat motif is (ATC) 6-8 ;

[0080] The Df06 is a DNA sequence shown in SEQ ID NO.06 or its complementary sequence, and its microsatellite repeat motif is (TG) 6-9;

[0081] The Df07 is a DNA sequence shown in SEQ ID NO.07 or its complementary sequence, and its microsatellite repeat motif is (ATG) 5-9 ;

[0082] The Df08 is a DNA sequence shown in SEQ ID NO.08 or its complementary sequence, and its microsatellite repeat motif is (GT) 7-10 ;

[0083] The Df09 is a DNA sequence shown in SEQ ID NO.09 or its complementary sequence, and its microsatellite repeat motif is (ATG) 4-5 ;

[0084] The Df10 is a DNA sequence shown in SEQ ID NO.10 or its complementary sequence, and its microsatellite repeat motif is (CAA) 5-7 ;

[0085] The Df11 is a DNA sequence shown in SEQ ID NO.11 or its complementary sequence, and its microsatellite repeat motif is (TTG) 6-9 ;

[0086] The Df12 is a DNA sequence shown in SEQ ID NO.12 or its complementary sequence, and its microsatellite repeat motif is (CA) 7-8 .

[0087] The present invention also provides a primer for a microsatellite marker of dust mites, wherein the primer comprises at least one of the following 12 pairs of primers, and the 12 pairs of primers are respectively:

[0088] The upstream primer Df01F is shown in SEQ ID NO.13; the downstream primer Df01R is shown in SEQ ID NO.14;

[0089] The upstream primer Df02F is shown in SEQ ID NO.15; the downstream primer Df02R is shown in SEQ ID NO.16;

[0090] The upstream primer Df03F is shown in SEQ ID NO.17; the downstream primer Df03R is shown in SEQ ID NO.18;

[0091] The upstream primer Df04F is shown in SEQ ID NO.19; the downstream primer Df04R is shown in SEQ ID NO.20;

[0092] The upstream primer Df05F is shown in SEQ ID NO.21; the downstream primer Df05R is shown in SEQ ID NO.22;

[0093] The upstream primer Df06F is shown in SEQ ID NO.23; the downstream primer Df06R is shown in SEQ ID NO.24;

[0094] The upstream primer Df07F is shown in SEQ ID NO.25; the downstream primer Df07R is shown in SEQ ID NO.26;

[0095] The upstream primer Df08F is shown in SEQ ID NO.27; the downstream primer Df08R is shown in SEQ ID NO.28;

[0096] The upstream primer Df09F is shown in SEQ ID NO.29; the downstream primer Df09R is shown in SEQ ID NO.30;

[0097] The upstream primer Df10F is shown in SEQ ID NO.31; the downstream primer Df10R is shown in SEQ ID NO.32;

[0098] The upstream primer Df11F is shown in SEQ ID NO.33; the downstream primer Df11R is shown in SEQ ID NO.34;

[0099] The upstream primer Df12F is shown as SEQ ID NO.35; the downstream primer Df12R is shown as SEQ ID NO.36.

[0100] The details are as follows:

[0101]

[0102] The FAM, HEX, ROX, and TAMRA mentioned in the above table are types of fluorescent dyes, and their Chinese meanings are 6-carboxyfluorescein, hexachloro-6-methylfluorescein, 6-carboxy-X-rhodamine, and 6-carboxytetramethylrhodamine, respectively.

[0103] The present invention also provides at least one application of the above-mentioned microsatellite marker or primer in the detection of population genetic diversity and genetic differentiation of dust mites.

[0104] The microsatellite primers provided in this embodiment have the characteristics of stable PCR amplification and high polymorphism, and can be used to analyze the genetic diversity and genetic structure of dust mites, which has high application value. It can make up for the shortcomings of microsatellite marker technology in the study of species such as dust mites, and provide support for the study of its application in genetic diversity and genetic differentiation analysis.

[0105] The present invention also provides a method for obtaining and analyzing dust mite primers, comprising the following steps:

[0106] Step 100, extracting dust mite DNA and sequencing after testing, the sequencing strategy is Illumina PE150, and the total sequencing read length is 300bp;

[0107] Step 200: Using the existing NCBI complete genome sequence of Dermatophagoides farinae as a reference gene, the sequencing data is compared using the BWA software, and after correction by the BestPractices process of GATK, the microsatellite loci are searched using the MISA software, polymorphic microsatellite markers are screened, and primers are designed. The specific parameters specified for the MISA software for microsatellite identification analysis are:

[0108] A single nucleotide with a single base repeating more than 12 times, a dinucleotide repeating 6 times or more, a trinucleotide or tetranucleotide repeating 5 times or more, and this type of repeat sequence is a microsatellite sequence;

[0109] Step 300: Screening out microsatellite markers with an allele greater than 3 from all obtained microsatellite markers based on high-throughput sequencing data of multiple sample genomes;

[0110] Step 400: Design primers for the flanking sequences of microsatellite markers using Primer 5 software; wherein the standard for primer design in step 400 is:

[0111] 1) Primer length is 15-30 bp, and the GC content of primers is between 40% and 60%;

[0112] 2) The length of the PCR amplified product is between 100-300 bp;

[0113] 3) The primer base distribution is random, and no more than 4 single bases should appear consecutively;

[0114] 4) The primer does not contain self-complementary sequence;

[0115] 5) There are no more than 4 complementary or homologous bases between a pair of primers;

[0116] 6) There is no complementarity or low complementarity between the forward and reverse primers.

[0117] Step 500: Perform PCR amplification using genomic DNA from different dust mite individuals as templates to obtain primers, and perform capillary electrophoresis detection to verify the microsatellite marker primers with strong specificity, good stability, and high polymorphism. The total volume of the PCR amplification reaction is 12.5 μl, including 6.25 μl 2×Taq PCR Mastermix, 0.5 μl template, and 0.5 μl of upstream and downstream primers, and add sterilized deionized water to make up to 12.5 μl;

[0118] Mix the PCR products evenly and place them in a PCR instrument. Pre-denature at 94°C for 5 min, denature at 94°C for 30 s, use a temperature gradient (51°C, 54°C, 57°C, 60°C) to test the optimal annealing temperature, extend at 72°C for 1 min 30 s, cycle 34 times, and finally extend at 72°C for 10 min.

[0119] After the reaction is completed, 1% agarose gel is used to screen the PCR amplification product whose length is in line with the expectation and the primers with a single band are temporarily stored at 4 degrees Celsius.

[0120] Example:

[0121] 1) The DNA of 1,000 dust mites was extracted using the phenol-chlorine method, and the extracted genomic DNA of dust mites was temporarily stored in a -20°C refrigerator. After passing the test, it was sequenced using the Illumina HiSeqTM platform. The sequencing strategy was IlluminaPE150, and the total sequencing read length was 300bp.

[0122] 2) Using the existing NCBI complete genome sequence of Dermatophagoides farinae as the reference gene, the sequencing data were aligned using the BWA software, and after correction by the BestPractices process of GATK, the software MISA (http: / / pgrc.ipk-gatersleben.de / misa / misa.html) was used for microsatellite identification analysis. The software specifies specific parameters, i.e., single nucleotides with more than 12 base repeats, dinucleotides with 6 or more base repeats, and trinucleotides and tetranucleotides with 5 or more base repeats. The sequences of this repeat type are microsatellite sequences. The screening results are as follows:

[0123] A total of 266 microsatellite markers were screened in this study.

[0124] Among them, trinucleotides are the most numerous, totaling 118, accounting for 44.36% of the total, followed by mononucleotides, totaling 98, accounting for 36.84% of the total, dinucleotide repeats totaling 43, accounting for 16.17%, and tetranucleotide repeats totaling 7, accounting for 2.64%.

[0125] From the analysis of the motif composition of microsatellite markers, among the four types of repeats, there are only A / T repeat units in the mononucleotide, with a total of 98. There are three types of dinucleotides, TC / GA, AT / AT, and AC / GT. Among them, AC / GT accounts for 74.42% of the total number of dinucleotide repeats, which is the most frequent dinucleotide sequence, followed by AT / AT, accounting for 13.95%. There are the most types of trinucleotides, with the largest number of ATG / CAT repeats, accounting for 50% of the total number of trinucleotide repeats, followed by GTT / AAC, accounting for 33.86%, AAT / ATT accounting for 11.86%, CTG / CAG accounting for 3.39%, TCG / CGA and TGG / CAA each with only one, accounting for 0.85% each. Among the tetranucleotide repeats, TTCA / TGAA has the largest number, but only three, accounting for 28.57% of the total number of tetranucleotide repeats, followed by ATTG / CAAT with two, accounting for 28.57%, TTTG / CAAA and TTTC / GAAA each with only one, accounting for 14.29% each. Among the microsatellite loci of D. farinae, A / T appeared most frequently, followed by ATG / CAT.

[0126] 3) Screening sites and primer design: From 266 microsatellite markers, 50 microsatellite markers with alleles greater than 3 were screened based on high-throughput sequencing data of multiple sample genomes.

[0127] 4) Primer design was performed based on the flanking sequences of microsatellite markers using Primer 5 software.

[0128] Primer design criteria are as follows:

[0129] ① The length is 15-30bp, and the GC content of the primer is between 40%-60%.

[0130] ②The length of the PCR amplification product is between 100-300bp.

[0131] ③ The distribution of bases is random, and no more than 4 single bases should appear consecutively.

[0132] ④The primer does not contain self-complementary sequence.

[0133] ⑤ There are no more than 4 complementary or homologous bases between a pair of primers.

[0134] ⑥ There is no complementarity or low complementarity between forward and reverse primers.

[0135] 5) PCR amplification: PCR amplification was performed using genomic DNA from different dust mite individuals as templates. The total volume of the PCR reaction was 12.5 μl, including 6.25 μl 2×Taq PCR Mastermix, 0.5 μl template, and 0.5 μl of upstream and downstream primers, and sterilized deionized water was added to make up to 12.5 μl. The PCR products were mixed evenly and placed in a PCR instrument, pre-denatured at 94°C for 5 min, denatured at 94°C for 30 s, and the optimal annealing temperature was tested using a temperature gradient (51°C, 54°C, 57°C, 60°C), extended at 72°C for 1 min 30 s, cycled 34 times, and finally extended at 72°C for 10 min. After the reaction was completed, 1% agarose gel was used to screen out 27 primers whose PCR amplification product length was in line with the expected length and had a single band, and they were temporarily stored at 4 degrees Celsius.

[0136] 6) Capillary electrophoresis detection: Capillary electrophoresis was performed using an ABI3730 sequencer from Anhui General Biosystems (Anhui) Co., Ltd. The polymorphism size was determined based on the number of alleles in the amplified product of each site and the results were screened.

[0137] 7) After PCR verification and polymorphic primer screening, 12 pairs of microsatellite primers with polymorphism, stable amplification, and clear and single bands were finally obtained. The 12 pairs of polymorphic primers were 20-25bp in size, and when the annealing temperature was 60°C, the target fragments were all amplified, with a length of about 166-276bp, which was in line with the design principles of microsatellite primers.

[0138] Based on the primers obtained above, this embodiment provides a method for analyzing population genetic diversity, and the specific steps are as follows:

[0139] (1) DNA extraction and PCR amplification

[0140] 1) Extract genomic DNA of D. farinae from three populations, and use the 12 pairs of fluorescently labeled microsatellite primers to perform PCR amplification on DNA templates of different D. farinae individuals.

[0141] 2) The PCR reaction system is as follows:

[0142] ①MIX12.5μL

[0143] ②10μM upstream and downstream primers 1μL each

[0144] ③DNA template 2μL

[0145] ④Add ultrapure water to 25 μL.

[0146] 3) The PCR amplification reaction conditions are:

[0147]

[0148] PCR products were stored at 4°C

[0149] (2) Capillary electrophoresis analysis: The PCR products of step (3) were subjected to genotyping analysis using capillary electrophoresis, and the genotype was determined based on the allele size of the amplified product at each locus. The length of the allele at each locus of each individual was read and analyzed using GeneMarker 2.2 software. The length of the allele fragment at each locus was counted using an excel spreadsheet, and the loci that failed to be read were recorded as 0.

[0150] 1) Genetic diversity analysis: Based on the genotyping data, the genetic diversity indicators of the geographical population of D. farinae were calculated using GenAlEx 6.502, including the number of alleles Na, the number of effective alleles Ne, the observed heterozygosity Ho, the expected heterozygosity He, and the Shannon index I. The polymorphism information content PIC of each locus was calculated using Cervus3.0.

[0151] 2) The number of alleles and polymorphism information content of each locus after amplification are shown in Table 2. The results showed that the number of alleles Na of these 12 loci in 15 dust mites ranged from 3 to 9, and the Df08 locus had the largest number of alleles, totaling 9; the locus with the highest number of effective alleles was also Df08, whose effective allele number Na was 4.840, and the effective allele number Ne of each locus ranged from 1.411 to 4.840. The observed heterozygosity Ho value ranged from 0.133 to 0.800. The expected heterozygosity He value of each locus ranged from 0.291 to 0.793; the Shannon index I of each locus ranged from 0.563 to 1.807. The polymorphism information content PIC of each locus ranged from 0.271 to 0.765. Except for loci Df05, Df10, Df11, and Df12, the other loci were greater than 0.5, showing high polymorphism and high genetic diversity.

[0152] The results of polymorphism analysis are shown in the following table:

[0153]

[0154]

[0155] The above embodiments are only exemplary embodiments of the present application and are not intended to limit the present application. The protection scope of the present application is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present application within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present application. Sequence Listing <110> Wannan Medical College <120> A dust mite microsatellite marker, primers and applications thereof, and a method for obtaining the primers <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 163 <212> DNA <213> Artificial Sequence <400> 1 aatataatca ctaatcagct tatgtttaac tctatttttg ccaactaaat ggtcaaagat 60 tatcaacacc atccctctat attattacag agcagaaaaa aaaacttgac gcatggcaaa 120 tattaattcg ttcgtggttt attgctactg ctgctgctgc tgc 163 <210> 2 <211> 151 <212> DNA <213> Artificial Sequence <400> 2 tctgtgttcg tctctttcat ggacgaatta attaacaaac aatgaacgaa ataataaacg 60 gttaaattct ctaaaaaccg aattttcgag tgaatgatga tgatgatgag gatgataaag 120 caacagaaaa aaaatttaat taatgcgaaa t 151 <210> 3 <211> 232 <212> DNA <213> Artificial Sequence <400> 3 ataaagttca gaccttcaat caatcaaata aaaaatgatc atcatcatca tcatcatcat 60 catcattttt aaataaacaa acaaaaacaa aatattttca aagaaaaaaa aatcaagaca 120 ttgataatga tgattagatt gacgataatt ttaattacaa attagctttt atttcgaatc 180 aaggtctcac taaattcttt gttaaaaaaa aataaaataa aatattcgaa cg 232 <210> 4 <211> 221 <212> DNA <213> Artificial Sequence <400> 4 attttttggt tgttgttgtt gttgttgttg atcaattaat aattacttaa tgctttgacc 60 ggaatggaaa tttgtcaaca agtgatgatt gaatatataa atgacataca taaatatgcc 120 ggatccatca atccattttg ttgttgtaaa gaatttgaca tcatcattgt tgatttataa 180 agaaaaaaac aaatttttta acttaatttt tcattcgttt t 221 <210> 5 <211> 172 <212> DNA <213> Artificial Sequence <400> 5 ttgtatcgtt gttatacatc cattaccaat tatacaatca ccatttggta ttatcaatga 60 ttcattatta tccgtatggt gatgattaca attttgaaca ataatcatca tcattatcat 120 tattagttcg atcatcatca tcatcatcat catcattggg tatggaaatt aa 172 <210> 6 <211> 174 <212> DNA <213> Artificial Sequence <400> 6 ttgatcaatc aatgaattat gaataataag gtatggatca ttcatcctaa tccaatgatt 60 atgtgtgtgt gtgtgtgtgt ttttgtttct cacaagattt ttttcgtgat tttctttttt 120 tgattcattg aacaatcatc agaagaaaaa aaaatcttaa agattcaatt ctta 174 <210> 7 <211> 150 <212> DNA <213> Artificial Sequence <400> 7 aatctaattt agcaacaacg aaaaaagttt gtttttcaaa gcggctatta atgtgatgat 60 aataattcca ttattttgta tttgtatcga atgatgtgta taaatggaaa tgatgatgat 120 gatggtgatg atgaaaaatt gaattgataa 150 <210> 8 <211> 181 <212> DNA <213> Artificial Sequence <400> 8 aaccaaccac caccaccacc accaccaacg acaaaaaaca taatcataat ctttgattat 60 atatcgtata tataagaaaa tatcaaatca tcatgggatg cggctaaaat aaaaaaacaa 120 aaaaagtttt tttatccgat tctgaatata ataaatccag ttagtgtgtg tgtgtgtcgg 180 t 181 <210> 9 <211> 226 <212> DNA <213> Artificial Sequence <400> 9 attataaaga ttttgagaga aaaaaaaata aagaaaatgt gatgaaaatg ccaaagaaga 60 atttctttgt catgttaatt ataatttgcc atttgttacc ttaaaatatt attaatttag 120 atgagctttg gatggatttt catcattttt tttctgtgtt ctaatttcta tgttttcatt 180 ttctccaaat tttttgatca ataatgatga tgatgatgaa gtaaac 226 <210> 10 <211> 202 <212> DNA <213> Artificial Sequence <400> 10 tttataaaat tcataaaatc tatttaaaaa tgatgaaaaa tttcgaataa acattattca 60 attcaacaac aacaacaata aaaagtcaca cttacctaag ttgaaataac atttaggaac 120 caaataacaa taacaatgaa tttaattaaa tcaacaagta tatggggaaa aagtgaaaat 180 caaaacaatg aaattaatgt tt 202 <210> 11 <211> 126 <212> DNA <213> Artificial Sequence <400> 11 ttcgtcgcta tgatgcgaca tttttgttgt tgttgttgtt gttgttgtcg atgaagattt 60 tctttttaaa cacaaacact tgttatctga ttattatgat gaggaaaaga aaaaaaaatt 120 aaatcc 126 <210> 12 <211> 179 <212> DNA <213> Artificial Sequence <400> 12 taatttcttc tctttttttt tatgtggctg ttggatacat cacacacaca cacacataca 60 aatataccgg acatactttg ttcatcaatc atttattcga aataaaaaac cggcaaaaaa 120 ataatcattt ttgattgcat catttttttt attcgcatcc ataaatcatc acgtgtaca 179 <210> 13 <211> twenty one <212> DNA <213> Artificial Sequence <400> 13 tgtgtgtgtg tgtgctgtag t 21 <210> 14 <211> twenty three <212> DNA <213> Artificial Sequence <400> 14 tgacggtgac caataaatac cca 23 <210> 15 <211> twenty one <212> DNA <213> Artificial Sequence <400> 15 tgtttcggac gacattgtgt g 21 <210> 16 <211> twenty one <212> DNA <213> Artificial Sequence <400> 16 gctgtccaaa atcacatggc a 21 <210> 17 <211> twenty four <212> DNA <213> Artificial Sequence <400> 17 tcaccatcat catctgtcaa atca 24 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <400> 18 tgtgagatgg gcacaacgaa 20 <210> 19 <211> twenty three <212> DNA <213> Artificial Sequence <400> 19 tcccaatgat tttcgttgtc gtc 23 <210> 20 <211> twenty two <212> DNA <213> Artificial Sequence <400> 20 accatcattc atccaccgat ca 22 <210> twenty one <211> 20 <212> DNA <213> Artificial Sequence <400> twenty one gttgttgtcg ctgctgaaca 20 <210> twenty two <211> twenty one <212> DNA <213> Artificial Sequence <400> twenty two acgttcaacc atacagcagg a 21 <210> twenty three <211> twenty two <212> DNA <213> Artificial Sequence <400> twenty three tcccctctgc tcaaatgaat gt 22 <210> twenty four <211> twenty four <212> DNA <213> Artificial Sequence <400> twenty four tggtaccgat tcttcaatca actt 24 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <400> 25 tctttctctg cgtgtgtgct 20 <210> 26 <211> twenty two <212> DNA <213> Artificial Sequence <400> 26 acggaaatat agctggggac ag 22 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <400> 27 tttgagctca ccaaccagcc 20 <210> 28 <211> 25 <212> DNA <213> Artificial Sequence <400> 28 ggcatcattt gattctgtat gctga 25 <210> 29 <211> twenty two <212> DNA <213> Artificial Sequence <400> 29 tcggcaattt caacacttgt gt 22 <210> 30 <211> twenty two <212> DNA <213> Artificial Sequence <400> 30 gcaaaacctt tgtagcagac ga 22 <210> 31 <211> twenty four <212> DNA <213> Artificial Sequence <400> 31 tcgtatgtgg cgataatttt gtcg 24 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <400> 32 ccggtgtatg cgtggctata 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <400> 33 gacgttgccg atggtattgc 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <400> 34 catgcaaagc agcacacgta 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <400> 35 tcacctcgat cacggacaaa 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <400> 36 tcgtgaccag accaatgtca 20

Claims

1. A primer for a microsatellite marker of Dermatophagoides farinae, characterized in that: The microsatellite marker is selected from at least one of Df01, Df02, Df03, Df04, Df06, Df07, Df08, and Df09; wherein, The Df01 is a DNA sequence shown in SEQ ID NO.01 or its complementary sequence, and its microsatellite repeat motif is (CTG) 5-8 ; The Df02 is a DNA sequence shown in SEQ ID NO.02 or its complementary sequence, and its microsatellite repeat motif is (ATG) 5-7 ; The Df03 is a DNA sequence shown in SEQ ID NO.03 or its complementary sequence, and its microsatellite repeat motif is (ATC) 9-12 ; The Df04 is a DNA sequence shown in SEQ ID NO.04 or its complementary sequence, and its microsatellite repeat motif is (GTT) 6-7 ; The Df06 is a DNA sequence shown in SEQ ID NO.06 or its complementary sequence, and its microsatellite repeat motif is (TG) 6-9 ; The Df07 is a DNA sequence shown in SEQ ID NO.07 or its complementary sequence, and its microsatellite repeat motif is (ATG) 5-9 ; The Df08 is a DNA sequence shown in SEQ ID NO.08 or its complementary sequence, and its microsatellite repeat motif is (GT) 7-10 ; The Df09 is a DNA sequence shown in SEQ ID NO.09 or its complementary sequence, and its microsatellite repeat motif is (ATG) 4-5 ; The primers include at least one of the following primers, and the primers are: The upstream primer Df01F is shown in SEQ ID NO.13; the downstream primer Df01R is shown in SEQ ID NO.14; The upstream primer Df02F is shown in SEQ ID NO.15; the downstream primer Df02R is shown in SEQ ID NO.16; The upstream primer Df03F is shown in SEQ ID NO.17; the downstream primer Df03R is shown in SEQ ID NO.18; The upstream primer Df04F is shown in SEQ ID NO.19; the downstream primer Df04R is shown in SEQ ID NO.20; The upstream primer Df06F is shown in SEQ ID NO.23; the downstream primer Df06R is shown in SEQ ID NO.24; The upstream primer Df07F is shown in SEQ ID NO.25; the downstream primer Df07R is shown in SEQ ID NO.26; The upstream primer Df08F is shown in SEQ ID NO.27; the downstream primer Df08R is shown in SEQ ID NO.28; The upstream primer Df09F is shown as SEQ ID NO.29; the downstream primer Df09R is shown as SEQ ID NO.

30.

2. Use of the primers according to claim 1 in detecting population genetic diversity and / or analyzing genetic differentiation of dust mites.

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