CN113817838A

CN113817838A - Dust mite microsatellite marker, primer and application thereof, and primer acquisition method

Info

Publication number: CN113817838A
Application number: CN202111013054.8A
Authority: CN
Inventors: 孙恩涛; 刘艺; 方惟希; 李咏琪; 王慧慧; 李梦楠; 王铎; 黄庆宇
Original assignee: Wannan Medical College
Current assignee: Wannan Medical College
Priority date: 2021-08-31
Filing date: 2021-08-31
Publication date: 2021-12-21

Abstract

The invention discloses a dust mite microsatellite marker, which 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 complementary sequences thereof in sequence, and microsatellite repeated motifs are (CTG) in sequence_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)_6‑9And (CA)_7‑8. The microsatellite marker and the primer thereof provided by the invention have the characteristics of stable PCR amplification, high polymorphism and the like, and can be used for carrying out genetic diversity and genetic knot of dust mitesThe structure analysis has higher application value.

Description

Dust mite microsatellite marker, primer and application thereof, and primer acquisition method

Technical Field

The invention relates to the technical field of molecular biology DNA marking, in particular to a dust mite microsatellite marker, a primer thereof, application thereof and a primer acquisition method.

Background

Dust mites belong to the family of dermatophagoides farinae without valve stocks, have wide habitat and are often bred in house dust, air conditioner dust-proof nets, flour, biscuit powder, animal feed, traditional Chinese medicinal materials, waste cotton and the like. The domestic distribution is mainly in the south and middle areas of China. Dust mites are commonly present in indoor environments where people live and work, and excrement, metabolites, sloughed off shells and dead mite bodies of the dust mites are strong allergens, and can cause mite allergic diseases, mite dermatitis, mite diseases in human bodies and the like.

At present, no specific medicine exists for mite allergic diseases caused by dust mite allergens, the number of dust mites in the environment is effectively controlled, and the effective prevention and treatment effects on the allergic diseases are achieved. And the research on the population genetics of the dust mites can provide data support for formulating a prevention strategy of the dust mites. Currently, few studies on the genetics of dust mite populations are being made, mainly due to the lack of effective molecular markers.

Microsatellite molecular markers (also called Simple Sequence Repeats (SSRs)) are widely distributed in eukaryotic genomes and consist of tandem repeat segments of 2-6 nucleotides, wherein double nucleotides (CA/GT) n is the most common in animals. Compared with other molecular markers, the microsatellite molecular marker has the advantages of large quantity, high polymorphism, good stability and repeatability, genetic codominance and the like, and is one of the molecular markers widely applied in recent years.

Therefore, the research on the genetic polymorphism and genetic differentiation of the dust mite population can be rapidly and accurately carried out by researching the polymorphic microsatellite marker, and data support is provided for formulating a prevention strategy of the dust mites.

Disclosure of Invention

The invention aims to provide a dust mite microsatellite marker, a primer thereof, application and a primer acquisition method, so as to solve the technical problems in the prior art.

In order to solve the technical problems, the invention specifically provides the following technical scheme:

the invention provides a dust mite microsatellite marker, which is selected from at least one of Df01, Df02, Df03, Df04, Df05, Df06, Df07, Df08, Df09, Df10, Df11 and Df 12; wherein,

the Df01 is a DNA sequence shown in SEQ ID NO.01 or a complementary sequence thereof, and the microsatellite repetitive motif is (CTG)_5-8；

The Df02 is a DNA sequence shown in SEQ ID NO.02 or a complementary sequence thereof, and the microsatellite repetitive motif is (ATG)_5-7；

The Df03 is a DNA sequence shown in SEQ ID NO.03 or a complementary sequence thereof, and the microsatellite repetitive motif is (ATC)_9-12；

The Df04 is a DNA sequence shown in SEQ ID NO.04 or a complementary sequence thereof, and the microsatellite repetitive motif is (GTT)_6-7；

The Df05 is a DNA sequence shown in SEQ ID NO.05 or a complementary sequence thereof, and the microsatellite repetitive motif is (ATC)_6-8；

The Df06 is a DNA sequence shown in SEQ ID NO.06 or a complementary sequence thereof, and the microsatellite repetitive motif is (TG)_6-9；

The Df07 is a DNA sequence shown in SEQ ID NO.07 or a complementary sequence thereof, and the microsatellite repetitive motif is (ATG)_5-9；

The Df08 is a DNA sequence shown in SEQ ID NO.08 or a complementary sequence thereof, and the microsatellite repetitive motif is (GT)_7-10；

The Df09 is a DNA sequence shown in SEQ ID NO.09 or a complementary sequence thereof, and the microsatellite repetitive motif is (ATG)_4-5；

The Df10 is a DNA sequence shown in SEQ ID NO.10 or a complementary sequence thereof, and the microsatellite repetitive motif is (CAA)_5-7；

The Df11 is shown in SEQ ID NO.11A DNA sequence or its complement with a microsatellite repeat motif of (TTG)_6-9；

The Df12 is a DNA sequence shown in SEQ ID NO.12 or a complementary sequence thereof, and the microsatellite repetitive motif is (CA)_7-8。

The invention also provides a dust mite microsatellite marked primer, which comprises at least one of the following 12 pairs of primers, wherein the 12 pairs of primers are respectively as follows:

the upstream primer Df01F is shown as SEQ ID NO. 13; the downstream primer Df01R is shown as SEQ ID NO. 14;

the upstream primer Df02F is shown as SEQ ID NO. 15; the downstream primer Df02R is shown as SEQ ID NO. 16;

the upstream primer Df03F is shown as SEQ ID NO. 17; the downstream primer Df03R is shown as SEQ ID NO. 18;

the upstream primer Df04F is shown as SEQ ID NO. 19; the downstream primer Df04R is shown as SEQ ID NO. 20;

the upstream primer Df05F is shown as SEQ ID NO. 21; the downstream primer Df05R is shown as SEQ ID NO. 22;

the upstream primer Df06F is shown as SEQ ID NO. 23; the downstream primer Df06R is shown as SEQ ID NO. 24;

the upstream primer Df07F is shown as SEQ ID NO. 25; the downstream primer Df07R is shown as SEQ ID NO. 26;

the upstream primer Df08F is shown as SEQ ID NO. 27; the downstream primer Df08R is shown as 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;

the upstream primer Df10F is shown as SEQ ID NO. 31; the downstream primer Df10R is shown as SEQ ID NO. 32;

the upstream primer Df11F is shown as SEQ ID NO. 33; the downstream primer Df11R is shown as SEQ ID NO. 34;

the upstream primer Df12F is shown as SEQ ID NO. 35; the downstream primer Df12R is shown in SEQ ID NO. 36.

The invention also provides at least one application of the microsatellite marker or the primer in population genetic diversity detection and genetic differentiation of the dust mites.

The invention also provides a method for acquiring the dust mite primer, which comprises the following steps:

step 100, extracting DNA of dust mites, detecting the DNA to be qualified, and then sequencing the DNA;

step 200, taking the existing NCBI dust mite whole genome sequence as a reference gene, comparing data obtained by sequencing by using BWA software, correcting by a BestPracts process of GATK, searching for a microsatellite marker by using software MISA, screening out the polymorphic microsatellite marker, and designing a primer.

Step 300, screening microsatellite markers with alleles larger than 3 from all obtained microsatellite markers according to high-throughput sequencing data of a plurality of sample genomes;

400, designing a Primer aiming at a flanking sequence of the microsatellite marker by using Primer 5 software;

and 500, carrying out PCR amplification by taking genome DNA of different dust mite individuals as templates to obtain primers, carrying out capillary electrophoresis detection, and verifying that the microsatellite marker primers with stronger specificity, good stability and high polymorphism are obtained through experiments.

In a preferred embodiment of the present invention, the sequencing strategy in step 100 is Illumina PE150, and the total sequencing read length is 300 bp.

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

the single nucleotide with 12 times or more of single base repetition, 6 times or more of two bases repetition, 5 times or more of three and four bases repetition, and the sequence of the repetition type is a microsatellite sequence.

As a preferred embodiment of the present invention, the design criteria of the primers in step 400 are:

1) the length of the primer is 15-30bp, and the GC content of the primer is 40-60 percent;

2) the length of the PCR amplification product is between 100-300 bp;

3) the distribution of the primer base is random, and more than 4 single bases do not continuously appear;

4) the primer does not contain self-complementary sequence;

5) there are no more than 4 complementary or homologous bases between a pair of primers;

6) the forward and reverse primers are not complementary or have low complementarity.

As a preferred scheme of the invention, the PCR reaction total volume of the PCR amplification is 12.5 μ l, and the PCR reaction total volume comprises 6.25 μ l of 2 xTaq PCR Mastermix, 0.5 μ l of template and 0.5 μ l of upstream and downstream primers respectively, and sterilized deionized water is added to make up to 12.5 μ l;

mixing PCR products uniformly and placing in a PCR instrument, performing pre-denaturation at 94 ℃ for 5min, performing denaturation at 94 ℃ for 30s, testing the optimal annealing temperature by using a temperature gradient, performing extension at 72 ℃ for 1min30s, circulating for 34 times, and performing final extension at 72 ℃ for 10 min;

after the reaction is finished, screening out a PCR product by using 1% agarose gel, and temporarily storing a primer with a single band and a length meeting the expectation at 4 ℃;

wherein the temperature gradient is 51 ℃, 54 ℃, 57 ℃ and 60 ℃.

As a preferred embodiment of the present invention, the size of the primer obtained in step 500 is 20-25bp, and the annealing temperature is 60 ℃, so that the target fragment can be amplified, and the length of the fragment is about 166-276 bp.

Compared with the prior art, the invention has the following beneficial effects:

1) the microsatellite marker and the primer thereof provided by the invention have the characteristics of stable PCR amplification, high polymorphism and the like, can be used for analyzing the genetic diversity and genetic structure of the dust mites, have higher application value, can make up the defects of the microsatellite marker technology in the research of the dust mite species, and provide support for the research of the application of the microsatellite marker technology in the genetic diversity and genetic differentiation analysis.

2) The invention compares the dust mite genome sequencing read data to NCBI dust mite reference genome, then utilizes MISA software to search and screen microsatellite loci, screens 50 microsatellite markers with polymorphism according to a plurality of sample genome high-throughput sequencing data, and further screens through a capillary electrophoresis experiment to finally obtain 12 microsatellite markers with high polymorphism and primers with high specificity and strong stability.

The development efficiency of the microsatellite marker is remarkably improved, and the defects of low analysis speed, complex operation, large error of quantitative result and low automation degree of conventional polyacrylamide gel electrophoresis and other methods can be overcome.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

FIG. 1 shows the fluorescence scan peak of the microsatellite locus fragment amplified by the first pair of primers

FIG. 2 shows the fluorescence scan peak of the microsatellite locus fragment amplified by the second primer pair

FIG. 3 shows the fluorescence scanning peak of the microsatellite locus fragment amplified by the third pair of primers

FIG. 4 shows the fluorescence scanning peak of the microsatellite locus fragment amplified by the fourth primer pair

FIG. 5 shows the fluorescence scanning peak of the microsatellite locus fragment amplified by the fifth primer pair

FIG. 6 shows the fluorescence scan peak of the microsatellite locus fragment amplified by the sixth pair of primers

FIG. 7 is a graph showing fluorescence scanning peaks of microsatellite locus fragments amplified by the seventh pair of primers

FIG. 8 shows the fluorescence scanning peak of the microsatellite locus fragments amplified by the eighth primer pair

FIG. 9 shows the fluorescence scanning peak of microsatellite locus fragments amplified by the ninth primer pair

FIG. 10 shows the fluorescence scanning peak of the microsatellite locus fragment amplified by the tenth primer pair

FIG. 11 is a graph showing fluorescence scanning peaks of microsatellite locus fragments amplified by the eleventh pair of primers

FIG. 12 shows the fluorescence scan peak of the microsatellite locus fragment amplified by the twelfth pair of primers.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The Df09 is a DNA sequence shown in SEQ ID NO.09Or the complement thereof, with a microsatellite repeat motif of (ATG)_4-5；

The Df11 is a DNA sequence shown in SEQ ID NO.11 or a complementary sequence thereof, and the microsatellite repetitive motif is (TTG)_6-9；

The method comprises the following specific steps:

FAM, HEX, ROX, TAMRA described in the above table are of the type of fluorescent dye, which means that in the order of Chinese, 6-carboxyfluorescein, hexachloro-6-methylfluorescein, 6-carboxy-X-rhodamine, 6-carboxytetramethylrhodamine.

The microsatellite primer provided by the embodiment has the characteristics of stable PCR amplification, high polymorphism and the like, can be used for analyzing the genetic diversity and the genetic structure of the dust mites, and has higher application value. The method can make up the defects of the microsatellite marker technology in the research of dust mite species, and simultaneously provides support for the research of the application of the microsatellite marker technology in genetic diversity and genetic differentiation analysis.

The invention also provides a dust mite primer obtaining and analyzing method, which comprises the following steps:

100, extracting DNA of dust mites, detecting the DNA to be qualified, and then sequencing, wherein the sequencing strategy is Illumina PE150, and the total sequencing read length is 300 bp;

step 200, taking the existing dust mite whole genome sequence of NCBI as a reference gene, comparing data obtained by sequencing by using BWA software, after the BestPracts process correction of GATK, searching microsatellite loci by using software MISA, screening polymorphic microsatellite markers and designing primers. (ii) a The specific parameters specified when the software MISA performs the microsatellite identification analysis are as follows:

single nucleotide with single base repetition times of more than 12, two bases repetition times of 6 times and more, three and four bases repetition times of 5 times and more, the repetitive sequence is a microsatellite sequence;

400, designing a Primer aiming at a flanking sequence of the microsatellite marker by using Primer 5 software; the design criteria of the primers in step 400 are as follows:

2) the length of the PCR amplification product is between 100-300 bp;

4) the primer does not contain self-complementary sequence;

And 500, carrying out PCR amplification by taking genome DNA of different dust mite individuals as templates to obtain primers, carrying out capillary electrophoresis detection, and verifying that the microsatellite marker primers with stronger specificity, good stability and high polymorphism are obtained through experiments. The total volume of PCR reaction for PCR amplification is 12.5. mu.l, and comprises 6.25. mu.l of 2 XTaq PCR Mastermix, 0.5. mu.l of template, and 0.5. mu.l of upstream and downstream primers, and sterilized deionized water is added to make up to 12.5. mu.l;

mixing PCR products, placing into a PCR instrument, pre-denaturing at 94 deg.C for 5min, denaturing at 94 deg.C for 30s, testing the optimal annealing temperature with temperature gradient (51 deg.C, 54 deg.C, 57 deg.C, 60 deg.C), extending at 72 deg.C for 1min30s, circulating for 34 times, and extending at 72 deg.C for 10 min;

after the reaction is finished, the length of the PCR amplified product is screened by using 1% agarose gel to be in accordance with the expectation, and the primer with a single band is temporarily stored at 4 ℃.

Example (b):

1) extracting 1000 dust mite DNA by using a phenol-chlorine method, placing the extracted dust mite genome DNA in a refrigerator at-20 ℃ for temporary storage, and sequencing by adopting an Illumina HiSeqTM platform after the detection is qualified. The sequencing strategy was Illumina PE150 with a total sequencing read length of 300 bp.

2) The existing dust mite whole genome sequence of NCBI is used as a reference gene, BWA software is used for comparing data obtained by sequencing, after the BestPracts process correction of GATK, software MISA (http:// pgrc.ipk-gatersleen.de/MISA. html) is used for carrying out microsatellite identification analysis, the software specifies specific parameters, namely single nucleotide with the number of single base repetition more than 12, two base repetition more than 6 times, three and four base repetition more than 5 times, and the sequence with the repetition type is a microsatellite sequence. The screening results were as follows:

a total of 266 microsatellite markers were selected in this study.

Wherein, the trinucleotide is maximum, 118 in total accounts for 44.36% of the total amount, the second one is mononucleotide, 98 in total accounts for 36.84% of the total amount, 43 in total accounts for 16.17% of dinucleotide repeats, and 7 in total accounts for 2.64% of tetranucleotide repeats.

From the microsatellite marker motif composition analysis, among these four repeat types, the mononucleotide has only A/T repeat units, for a total of 98. Dinucleotide-consensus TC/GA, AT/AT, AC/GT, among the three types, AC/GT accounts for 74.42% of the total number of dinucleotide repeats, is the most frequent dinucleotide sequence, and the second is AT/AT accounts for 13.95%. The types of trinucleotides are the most, the number of ATG/CAT repeats is the most, and accounts for 50% of the total number of trinucleotide repeats, the number of GTT/AAC repeats is 33.86%, AAT/ATT accounts for 11.86%, CTG/CAG accounts for 3.39%, and only one of TCG/CGA and TGG/CAA accounts for 0.85% respectively. In the tetranucleotide repeats, TTCA/TGAA counts the most, but only three, 28.57% of the total number of tetranucleotides, and secondly 2 ATTG/CAAT, 28.57%, and only one of TTTG/CAAA and TTTC/GAAA, 14.29% each. In the dust mite microsatellite locus, the A/T repeats occur most frequently, followed by ATG/CAT.

3) Screening site and primer design: from the 266 microsatellite markers, microsatellite markers with 50 alleles greater than 3 were selected based on high throughput sequencing data of a plurality of sample genomes.

4) Primer design was performed for the flanking sequences of the microsatellite marker using Primer 5 software.

The primer design criteria were as follows:

the length of the primer is 15-30bp, and the GC content of the primer is 40-60%.

② the length of the PCR amplified product is between 100 and 300 bp.

③ the distribution of the basic groups is random, and more than 4 single basic groups should not appear continuously.

And fourthly, the primer does not contain self-complementary sequence.

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

Sixthly, the complementarity does not exist between the forward primers and the reverse primers or the complementarity is low.

5) And (3) PCR amplification: and (3) carrying out PCR amplification by taking the genome DNA of different dust mite individuals as a template. The total volume of the PCR reaction was 12.5. mu.l, containing 6.25. mu.l of 2 XTAQQ PCR Mastermix, 0.5. mu.l of template, and 0.5. mu.l of each of the upstream and downstream primers, and was made up to 12.5. mu.l with sterilized deionized water. The PCR product was mixed well and placed in a PCR instrument, pre-denatured at 94 ℃ for 5min, denatured at 94 ℃ for 30s, tested for optimal annealing temperature using temperature gradients (51 ℃, 54 ℃, 57 ℃, 60 ℃), extended at 72 ℃ for 1min30s, cycled 34 times, and finally extended at 72 ℃ for 10 min. After the reaction is finished, 27 primers with the expected length and single band of the PCR amplified product are screened out by using 1% agarose gel and temporarily stored at 4 ℃.

6) And (3) capillary electrophoresis detection: capillary electrophoresis was performed by ABI3730 sequencer of general biosystems of Anhui (Anhui) Inc. Judging the size of the polymorphism according to the number of alleles of the amplification product of each site, and screening.

7) Through PCR verification and polymorphic primer screening, 12 pairs of dust mite microsatellite primers with polymorphism, stable amplification and clear and single bands are finally obtained. The size of the 12 pairs of polymorphic primers is 20-25bp, when the annealing temperature is 60 ℃, the target fragment can be amplified, the length of the fragment is about 166-276bp, and the design principle of the microsatellite primer is met.

Based on the primers obtained above, the present embodiment provides a method for analyzing genetic diversity of a population, which comprises the following specific steps:

(1) DNA extraction and PCR amplification

1) Extracting the genome DNA of the dust mites of 3 populations, and carrying out PCR amplification on DNA templates of different dust mite individuals by using the 12 pairs of fluorescence labeling microsatellite primers.

2) The PCR reaction system is as follows: are respectively as

①MIX12.5μL

10 mu M of upstream and downstream primers each 1 mu L

(iii) 2. mu.L of DNA template

Fourthly, supplying ultrapure water to 25 mu L.

3) The PCR amplification reaction conditions are as follows:

the PCR product was stored at 4 ℃

(2) Capillary electrophoresis analysis: and (4) carrying out genotyping analysis on the PCR product of the step (3) by using capillary electrophoresis, and determining the genotype according to the allele size of the amplification product of each site. The length of the allele at each locus of each individual was analyzed by GeneMarker 2.2 software reads. The length of the fragment for each site allele was counted using an excel table and recorded as 0 for sites that failed to be read.

1) Genetic diversity analysis: according to the genotyping data, genetic diversity indicators of the geographic population of dust mites were calculated using GenAlEx 6.502, including allele numbers Na, effective allele numbers Ne, observed heterozygosity Ho, expected heterozygosity He, Shannon index I. The polymorphism information content PIC of each site is calculated by using Cervus 3.0.

2) The allele counts and the polymorphic content of each locus after amplification are shown in Table 2. The results show that the allele factors Na of the 12 loci in 15 dust mites are between 3 and 9, and the number of alleles of the Df08 locus can be detected to be 9 at most; the locus with the highest number of effective alleles is Df08, the effective allele factor Na of the locus is 4.840, and the effective allele factors Ne of each locus are all between 1.411 and 4.840. The value of the observed heterozygosity Ho is between 0.133 and 0.800. The value of the desired heterozygosity He at each position is 0.291-0.793; the shannon index I of each site is between 0.563 and 1.807. The polymorphism information content PIC of each site is between 0.271 and 0.765, and except the sites Df05, Df10, Df11 and Df12, other sites are all larger than 0.5, show high polymorphism and have high genetic diversity.

The polymorphism analysis results are shown in the following table:

the above embodiments are only exemplary embodiments of the present application, and are not intended to limit the present application, and the protection scope of the present application is defined by the claims. Various modifications and equivalents may be made by those skilled in the art within the spirit and scope of the present application and such modifications and equivalents should also be considered to be within the scope of the present application.

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tgtgagatgg gcacaacgaa 20

<210> 19

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tcccaatgat tttcgttgtc gtc 23

<210> 20

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

accatcattc atccaccgat ca 22

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gttgttgtcg ctgctgaaca 20

<210> 22

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

acgttcaacc atacagcagg a 21

<210> 23

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

tcccctctgc tcaaatgaat gt 22

<210> 24

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

tggtaccgat tcttcaatca actt 24

<210> 25

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

tctttctctg cgtgtgtgct 20

<210> 26

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

acggaaatat agctggggac ag 22

<210> 27

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

tttgagctca ccaaccagcc 20

<210> 28

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ggcatcattt gattctgtat gctga 25

<210> 29

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

tcggcaattt caacacttgt gt 22

<210> 30

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

gcaaaacctt tgtagcagac ga 22

<210> 31

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

tcgtatgtgg cgataatttt gtcg 24

<210> 32

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

ccggtgtatg cgtggctata 20

<210> 33

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

gacgttgccg atggtattgc 20

<210> 34

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

catgcaaagc agcacacgta 20

<210> 35

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

tcacctcgat cacggacaaa 20

<210> 36

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

tcgtgaccag accaatgtca 20

Claims

1. A dust mite microsatellite marker selected from at least one of Df01, Df02, Df03, Df04, Df05, Df06, Df07, Df08, Df09, Df10, Df11 and Df 12; wherein,

the Df01 is a DNA sequence shown in SEQ ID NO.01 or a complementary sequence thereof, and the DNA sequence isMicrosatellite repetitive elements are (CTG)_5-8；

2. The primer marked by the dust mite microsatellite is characterized by comprising at least one of the following 12 pairs of primers, wherein the 12 pairs of primers are respectively as follows:

3. Use of the microsatellite marker according to claim 1 or the primer according to claim 2 for at least one of detecting genetic diversity and genetic differentiation of dust mites.

4. A method for obtaining a primer marked by a dust mite microsatellite is characterized by comprising the following steps:

step 200, taking the existing whole genome sequence of dust mite at NCBI as a reference gene, comparing data obtained by sequencing by using BWA software, correcting by a BestPracts process of GATK, searching for a microsatellite marker by using software MISA, screening out the polymorphic microsatellite marker in a dust mite genome, and designing a primer.

Step 300, screening out microsatellite markers with alleles larger than 3 from all obtained dust mite microsatellite markers according to high-throughput sequencing data of a plurality of sample genomes;

5. The method for obtaining the primer labeled with the dust mite microsatellite according to claim 4, wherein the sequencing strategy in the step 100 is Illumina PE150, and the total sequencing read length is 300 bp.

6. The method for obtaining primer of dust mite microsatellite marker as set forth in claim 4, wherein the specific parameters specified by software MISA in step 200 for microsatellite identification analysis are:

7. The method for obtaining primer of dust mite microsatellite marker as set forth in claim 4, wherein the design criteria of primer in step 400 are:

2) the length of the PCR amplification product is between 100-300 bp;

4) the primer does not contain self-complementary sequence;

8. The method for obtaining the primer of the dust mite microsatellite marker as set forth in claim 4, wherein the PCR amplification reaction has a total volume of 12.5 μ l, and comprises 6.25 μ l of 2 XTaq PCR Mastermix, 0.5 μ l of template, and 0.5 μ l of each of the upstream and downstream primers, and sterilized deionized water is added to make up to 12.5 μ l;

wherein the temperature gradient is 51 ℃, 54 ℃, 57 ℃ and 60 ℃.

9. The method for obtaining the primer labeled with the dust mite microsatellite according to claim 4, wherein the size of the primer obtained in step 500 is 20-25bp, and the annealing temperature is 60 ℃, so that the target fragment can be amplified, and the length of the fragment is about 166-276 bp.