CN106399497B - Pistacia chinensis bunge microsatellite loci, primers and application - Google Patents

Abstract

The invention aims to provide a pistacia chinensis bunge microsatellite locus, a primer and application. The invention provides 12 pistacia chinensis bunge microsatellite loci, and primers designed from the microsatellite loci are used for detecting the population genetic diversity of the pistacia chinensis bunge. According to the invention, 12 microsatellite loci are obtained from the Pistacia chinensis genome by using a magnetic bead enrichment method and a fluorescent marker sequencing technology, and specific primers are designed according to flanking sequences at two ends of the microsatellite, so that the amplification result has high polymorphism and stability, and can be used in the fields of Pistacia chinensis group genetic diversity detection, genetic relationship identification, genetic linkage map construction, molecular marker assisted breeding and the like.

Description

Pistacia chinensis bunge microsatellite loci, primers and application

Technical Field

The invention relates to the technical field of molecular biology DNA marking, in particular to a pistacia chinensis bunge microsatellite locus, a primer and application.

Background

Pistacia chinensis Bunge belonging to genus Pistacia of Anacardiaceae (Anacardiaceae), heterostrain, and deciduous tree. The Chinese goldthread is happy by yang and grows in families, has certain tolerance to drought and barrenness, is easy to cultivate, is widely distributed in China, and is recorded in 23 provinces and urban areas such as northwest, north China, east China, south China and the like. The Pistacia chinensis has high ornamental value and high economic development value, and the wood is tough and corrosion-resistant and belongs to high-grade building and furniture materials; the leaves, seeds, bark, etc. can be used as medicine; the oil content of the seeds is up to 35.05 percent, and the pistacia chinensis bunge seed oil not only can be used as edible oil, but also can be used as a raw material of products such as fatty acid, lubricating oil, soap and the like, and more importantly, is one of important sources of biodiesel and has been paid more and more attention and researched. As the pistacia chinensis bunge is an important source of renewable green energy biodiesel and has high ecological and economic values, researches on the pistacia chinensis bunge are more and more emphasized, but the researches on the pistacia chinensis bunge are mainly focused on the aspects of physiology, distribution, energy development and the like, and the researches on the aspects of molecular biological level, germplasm resource analysis, population genetic structure and the like are very few.

The Microsatellite (Microsatellite), also called Simple Sequence Repeat (SSR), is usually composed of a core Sequence with 1-6 nucleotides as a Repeat unit and a relatively conserved flanking Sequence, along with the development of molecular markers, the Microsatellite markers are widely applied to the research of population genetic structure and genetic relationship, genetic breeding, map construction, germplasm resource identification and the like as the characteristics of good genetic stability, rich information, strong universality, co-dominant inheritance and the like, the acquisition path of the Microsatellite primers can be roughly divided into the screening from closely related species of a published table, the Microsatellite loci are searched from the Sequences of the same closely related species in a public database, the Microsatellite loci are screened by a method for constructing a library from genomic DNA, the efficiency of the method for screening the Microsatellite loci by constructing the library is highest in the three methods, and the patent utilizes a method for constructing an AFCO (biological Fast — enrichment magnetic bead) enriched library based on AF 24P (Amplified Fragment length Polymorphism, Amplified Fragment L enhanced Polymorphism).

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a pistacia chinensis bunge microsatellite locus and a polymorphic primer, namely 12 pistacia chinensis bunge microsatellite loci and a corresponding polymorphic microsatellite primer are provided to provide a molecular marker for the identification of the validity of pistacia chinensis bunge species and the analysis of population genetic diversity.

The invention screens 6 microsatellite sequences from Pistacia chinensis genome DNA by a magnetic bead hybridization method, wherein the GenBank accession number is KT780354-365, and the microsatellite sequences are respectively shown as SEQ ID NO: 1 to 12.

Microsatellite sequences also include nucleotide sequence molecules comprising the same microsatellite repeat units which hybridize under stringent conditions to the nucleotide sequences described above.

The Pistacia chinensis microsatellite primers are forward and reverse primers designed from flanking sequences of microsatellite repeats of sequences PC1, PC19, PC44, PC64, PC70, PC96, PC106, PC172, PC189, PC193, PC196 and PC 205.

The sequences of the microsatellite primers are respectively SEQ ID NO: PC1F/R, PC19F/R, PC44F/R, PC64F/R, PC70F/R, PC96F/R, PC106F/R, PC172F/R, PC189F/R, PC193F/R, PC196F/R, PC 205F/R.

Microsatellite sites, site sequences and corresponding primer information are shown in table 1:

TABLE 1 basic characteristics of 12 pairs of primers obtained from Pistacia chinensis microsatellite primer development

The microsatellite polymorphism primer is used for detecting the genetic diversity of the Pistacia chinensis population, and comprises the following steps:

1) extraction of genomic DNA: extracting the genome DNA of the Pistacia chinensis by slightly adjusting the characteristics of the Pistacia chinensis on the basis of the operation of a plant leaf DNA extraction kit of Tiangen Biochemical technology (Beijing) Ltd;

2) micro-satellite PCR amplification: carrying out PCR by adopting designed primers and the Pistacia chinensis genome DNA as a template to obtain a Pistacia chinensis individual microsatellite amplification product;

3) detecting the amplification product by electrophoresis, namely detecting the microsatellite amplification product by adopting a modified polyacrylamide gel electrophoresis and silver staining method, and sequencing the amplification result by utilizing an ABI 3730X L sequencer;

4) genetic diversity analysis, determining the genotype according to the molecular weight of each individual microsatellite amplification product, carrying out population genetic analysis by utilizing POPGENE Version 1.32 software after the sequence result is judged by Genmapper4.0, calculating the allele factor (Na), the observed heterozygosity (Ho), the expected heterozygosity (He), whether the Hardy-Weinberg balance (P) deviates, and calculating the information content by utilizing PIC software (PIC _ CA L Version C0.6).

The invention screens 12 microsatellite loci from Pistacia chinensis genome DNA, designs specific primers according to flanking sequences of the loci at two ends of the microsatellite loci, has high polymorphism and stability of amplification results of the obtained primers, and can be used in the fields of Pistacia chinensis population genetic diversity detection, species validity identification, molecular assisted breeding and the like.

Drawings

FIG. 1 is a diagram showing the result of Pistacia chinensis genome agarose gel electrophoresis;

FIG. 2 shows the result of restriction enzyme digestion of Pistacia chinensis genome;

FIG. 3 shows the result of the amplification of the enriched products from the magnetic beads of Pistacia chinensis Bunge;

FIG. 4 is the PCR detection electrophoresis pattern of the positive clone of Pistacia chinensis Bunge.

Detailed Description

The present invention is further analyzed with reference to the following specific examples.

Genome DNA extraction of pistacia chinensis bunge

The invention extracts the Pistacia chinensis genome by slightly adjusting the characteristics of the Pistacia chinensis on the basis of the operation of a plant leaf DNA extraction kit of Tiangen Biochemical technology (Beijing) Co.

(1) Weighing about 30mg Pistacia chinensis Bunge silica gel, drying to remove petiole and leaf vein, placing in 2ml centrifuge tube, soaking in liquid nitrogen for 1min, and grinding in grinding instrument.

(2) Adding 700 ul of preheated GP1 and 2 ul of β -mercaptoethanol into the centrifuge tube, uniformly mixing by vortex oscillation, carrying out water bath at 65 ℃ for 50min, and reversing the centrifuge tube for a plurality of times in the water bath process to uniformly mix the sample.

(3) Add 700. mu.l chloroform, mix well and centrifuge at 12,000rpm for 5 min.

(4) The upper layer was transferred to a new centrifuge tube, 700. mu.l GP2 was added, vortexed and mixed well.

(5) The liquid was transferred to an adsorption column CB3, centrifuged at 12,000rpm for 30 seconds, and the waste liquid was discarded.

(6) CB3 was replaced in the collection tube, 500. mu.l of GD buffer was added, 12,000rpm was centrifuged for 30s, and the waste was discarded.

(7) To the adsorption column CB3, 600. mu.l of the rinsing solution was added, and the mixture was centrifuged at 12,000rpm for 30 seconds, and the waste solution was discarded. Repeat twice, and leave at room temperature for 15 min.

(8) Transferring CB3 into a sterile centrifuge tube, suspending and dropwise adding 100 μ l of ultrapure water into the middle part of the adsorption membrane, standing at room temperature for 5min to fully dissolve DNA, and centrifuging at 12,000rpm for 2 min. The centrifuged solution was resuspended in an adsorption column of CB3, left to stand for 10min, and centrifuged at 5,000rpm for 1min to obtain a DNA stock solution.

(9) The quality of the obtained genome was checked by electrophoresis using 1.5% agarose gel, and a sample with a clear band was selected, as shown in FIG. 1, and its quality and concentration were measured using a micro-UV spectrophotometer.

Second, genome DNA enzyme digestion and magnetic bead enrichment microsatellite library

1. Genomic DNA restriction, adaptor ligation and product pre-amplification

(1) And (3) carrying out genome enzyme digestion. Carrying out enzyme cutting by using restriction enzyme MseI, wherein the enzyme cutting system is 25 mu l: genomic DNA250ng, endonuclease 5U, Cutsmart 2.5. mu.l, ddH₂O, a replenishing system; water bath at 37 deg.C for 1h, and water bath at 65 deg.C for 20 min. The cleavage result was detected by 1.5% agarose gel electrophoresis, and the band having a size of 100-800bp was separated and recovered as shown in FIG. 2.

(2) And (4) connecting enzyme digestion products. The ligation reaction contained 30. mu.l total, containing 15. mu.l of enzyme cleavage product: t4DNA ligase 4.0U, MseI linker 30pmol (linker sequence F: 5'-TACTCAGGACTCAT-3', SEQ ID NO: 37, R: 5'-GACGATGAGTCCTGAG-3', SEQ ID NO: 38), ddH₂O is filled in the system and is connected for 12 hours at 16 ℃.

(3) The PCR amplification system is 20 ul containing 10 × Buffer 2 ul, dNTPs (2.5mM)5 ul, Taq DNA polymerase 0.5U, enzyme digestion ligation product 5 ul, MseI-N primer (5 '-GATGAGTCCTGAGTAAN-3') 20pmol PCR reaction program of pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 60s, extension at 72 ℃ for 60s, 20 cycles, extension at 72 ℃ for 8min, and preservation at 4 ℃.

2. Biotin probe and pre-amplification product hybridization and magnetic bead enrichment

(1) Marked with biotin (AG)₁₅For hybridization of probe and pre-amplification product, the hybridization reaction system was 250. mu.l, in which 30. mu.l of PCR product, 300pmol of probe, 52.5. mu.l of 20 × SSC, 1.75. mu.l of 10% SDS, ddH₂After the mixture is completely filled with O, the mixture is gently mixed, denatured at 95 ℃ for 5 minutes and then placed in a water bath at 48 ℃ for hybridization for 2 hours. And (3) mixing the processed magnetic beads with the hybridization products, keeping the temperature at room temperature for 50min, continuously and gently shaking the mixture, placing the reaction products on a magnetic rack for adsorption, and gently sucking out the liquid by using a pipette.

(2) Non-stringent washes. Wash 3 times with TEN 1000500 μ l, 8 min/time. After the treatment, the reaction product was placed on a magnetic stand for adsorption, and the liquid was gently aspirated out with a pipette.

(3) Stringent washing was performed 3 times using 500. mu.l of 0.2 × SSC +0.1% SDS.

(4) And (3) adding 50. mu.l of 1 × TE, carrying out water bath at 95 ℃ for 5min, placing the reaction product on a magnetic rack for adsorption, gently sucking out the liquid by using a pipette gun, and quickly placing the liquid into another sterile 1.5ml centrifugal tube.

(5) Amplification and purification of the enriched product was amplified with MseI-N primer (5 '-GATGAGTCCTGAGTAAN-3') in a total reaction system of 20. mu.l containing 10 × Buffer 2. mu.l, dNTPs (2.5mM) 2. mu.l, primer 20pmol, Taq DNA polymerase 0.5U, template 5. mu.l, ddH₂And O is used for supplementing the system. The PCR reaction program comprises pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 60s, extension at 72 ℃ for 60s, 20 cycles, extension at 72 ℃ for 8min, and storage at 4 ℃.

(6) The detection is carried out by using 1.5% agarose gel electrophoresis, and if the product presents a uniform dispersion band and the size is between 200 and 800bp, the enrichment elution process is more successful, as shown in FIG. 3. Then, the target product is recovered by using a PCR product purification kit.

2. Enriched product clone and positive clone detection

(1) And (4) connecting carriers. The purified product was ligated into pMD19-T vector in a ligation reaction system of 4.5. mu.l of purified DNA from the previous step, 0.5. mu.l of T vector, 5. mu.l of ligation solution, and 10. mu.l in total. The reaction program was 16 ℃ and ligation was performed for 2h without using a hot lid in the PCR instrument.

(2) And (4) converting a connection product. The ligated product was added to 100. mu.l of competent cells, allowed to stand on ice for 30min, then quickly transferred to a 42 ℃ water bath for 45 seconds, and then allowed to stand on ice for 1 min. Adding 890. mu.l SOC culture medium, and culturing at 37 deg.C under shaking for 60 min.

(3) And (3) coating the connected transformation product in L B solid culture medium containing 100 mg/L Amp, culturing overnight at 37 ℃, picking out full and bright positive clones, and culturing in L B liquid culture medium containing Amp at 250r/min and 37 ℃ until the bacterial liquid is slightly turbid.

(4) The restriction enzyme MseI was used to sequence the universal primer M13 (M13-47: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3';

m13-48: 5'-AGCGGATAACAATTTCACACAGGA-3') and the aforementioned probe (AG)15 without biotin labeling were used to perform PCR amplification on the enriched library (primer combinations are a: M13-47+ (AG) 15; b: M13-48+ (AG) 15; c: M13-47+ M13-48; screening positive clones, respectively.) the PCR system was 15. mu.l containing 10 × Buffer 1.5. mu.l, dNTPs (2.5mM) 2. mu.l, upstream and downstream primers each 30pmol, Taq DNA polymerase 0.25U, bacterial solution 2. mu.l, and ddH2O were supplemented, the PCR reaction program was pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, elongation at 72 ℃ for 30s, 30 cycles, elongation at 72 ℃ for 8min, and storage at 4 ℃ the amplification results were examined by 1.5% agarose gel electrophoresis, and sequencing the positive clones were sent to Hakko Biochemical engineering Co., Ltd, as shown in FIG. 4.

Third, microsatellite primer

The positive clone sequencing result utilizes DNA star 8.0 to remove a vector sequence, SSR Hunter 1.3 to search a core sequence, a sequence with a longer microsatellite flanking sequence and more repeated times of a repeating unit is selected, Primer premier5.0 is utilized to design a Primer, and the principle followed when the Primer is designed mainly has the Primer length of 18-22 bp; GC content is 40-60%; the Tm value is 45-65 ℃, and the optimal Tm value is 55 ℃; the GC content difference of the positive primer and the negative primer is within 10 percent, and the Tm value difference is within 5 ℃; the product length is 200 and 400 bp; the designed primers were synthesized by Shanghai Biotechnology Ltd.

Detection of validity and universality of microsatellite primers

1. Detection of the effectiveness of primers

The primers obtained by the primary screening are subjected to PCR amplification by using 24 Pistacia chinensis Bunge individuals, the amplification reaction system is 15 mu l, wherein the amplification reaction system comprises 1.5 mu l of 10 × Buffer, 1.5 mu l of dNTPs (2.5mM), 50pmol of each of upstream and downstream primers, 0.25U of Taq DNA polymerase, 1.5 mu l of template and completion of ddH2O, the PCR reaction program comprises the steps of pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at Tm (51-59 ℃) for 30s, extension at 72 ℃ for 30s, circulation for 30, extension at 72 ℃ for 8min and storage at 4 ℃.

And (3) detecting the polymorphism of the amplification result by using 8% (W/V) non-denaturing polyacrylamide gel electrophoresis (PAGE) and silver nitrate staining after photographing, and selecting the microsatellite primer with the polymorphism. The electrophoresis detection steps are as follows:

(1) groove installation: before the groove is filled, the inner sides of the two glass plates are wiped by 100% absolute ethyl alcohol, adhesive tapes for sealing the adhesive tapes are filled, then the two glass plates are fixed in an electrophoresis groove, and the two ends of the electrophoresis groove are clamped by a clamp.

(2) Sealing glue: the edges of the electrophoresis tank were sealed with agar at a concentration of 2% to prevent leakage of the gel.

(3) Gel preparation 8% PAGE gel formulation (100 ml total: deionized water 51.25 ml; 40% Acrylamide solution 20 ml; 10 × TBE solution 20 ml; 20% ammonium persulfate 1 ml; TEMED 100. mu.l).

(4) Glue pouring: after stirring uniformly, pouring into a filled glue groove, slightly and vertically inserting a comb, and standing for 2 hours at room temperature.

(5) Loading: and sequentially adding 10 mu l of PCR product mixed with the sample loading buffer solution into the sample adding hole, selecting 500bp DNAmarker and 200V, carrying out electrophoresis at constant voltage until the indicator reaches the bottom of the gel plate, and stopping electrophoresis.

(6) Silver staining developing and photographing record

① separating the rubber plate, washing the rubber plate with flowing water for separation, punching holes on the right side of the rubber plate for marking, and performing operation on a shaker in the following steps to make the effect more sufficient.

② rinsing, soaking the gel plate in 500ml distilled water for 5min, and removing the ions in the residual buffer solution on the surface.

③ dyeing, namely, placing the rubber plate in 1 percent AgNO3 dyeing solution to soak the rubber plate for at least 5min, wherein the time can be properly prolonged, and the effect is better.

④ washing, soaking the above gel plate in 500ml distilled water for 5min to remove the residual anions on the surface.

⑤ color development, preparing 500ml of mixed solution of 1 percent developing solution and 2ml of formaldehyde solution, quickly placing the rubber plate in the mixed solution after uniformly mixing, closely observing, taking out the rubber plate when a strip is hidden, placing the rubber plate in 500ml of distilled water to stop dyeing, and then photographing to record the result.

2. Primer commonality detection

The screened primers with effective polymorphism are labeled by 6' -FAM, HEX, TAMRA and ROX, PCR amplification is carried out by using 8 individuals in one continent population and two island populations of the Qiandao lake under the same amplification condition as a PAGE gel screening system and procedure except that the annealing temperature is increased by 2 ℃, the amplification result is sequenced by using an ABI 3730X L sequencer, the sequencing result is interpreted by Genmapper4.0 and then is subjected to genetic analysis by using POPGENE 1.32 software, relevant indexes such as microsatellite allelic factor (Na), observed heterozygosity (Ho), expected heterozygosity (He) and whether Hardy-Weinberg balance is deviated are calculated, and the content of polymorphic information is calculated by using PIC software (PIC _ CA L C0.6).

As shown in table 2, the total number of detected alleles in 24 individuals of the 12 pairs of primers obtained by screening was 125; the variation range of the allelic base factors of the single individuals is 2-8, and the average value is 3.5; the Polymorphic Information Contents (PIC) of the three populations are respectively 0.5449 (0.3589-0.7993), 0.4931 (0.3047-0.7654) and 0.5442 (0.1948-0.8157); observed heterozygosity (Ho) and expected heterozygosity (He) range from 0.1250 to 0.8750 and 0.2333 to 0.8917, with allele frequencies at 12 loci in accordance with Hardy-Weinberg equilibrium, except for the PC193 locus in the Nepeta and the PC205 locus in the continental population.

SEQUENCE LISTING

<110> Qianjiang college of Hangzhou university

<120> pistacia chinensis bunge microsatellite locus, primer and application

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ggttagtttg ctcgctct 18

<210>29

<211>21

<212>DNA

<213> Artificial Synthesis

<400>29

tgagtcctga gtaagcaaat g 21

<210>30

<211>20

<212>DNA

<213> Artificial Synthesis

<400>30

ttagagggcg tgaagatgta 20

<210>31

<211>20

<212>DNA

<213> Artificial Synthesis

<400>31

taaggatgtt tgtgtggagg 20

<210>32

<211>20

<212>DNA

<213> Artificial Synthesis

<400>32

taaggtttta ccaaatgcca 20

<210>33

<211>20

<212>DNA

<213> Artificial Synthesis

<400>33

cacggtatcc atctcccttt 20

<210>34

<211>20

<212>DNA

<213> Artificial Synthesis

<400>34

agtaaggtgg ttcggatgct 20

<210>35

<211>20

<212>DNA

<213> Artificial Synthesis

<400>35

aaagtatctg ttatgttggg 20

<210>36

<211>20

<212>DNA

<213> Artificial Synthesis

<400>36

tgagtcctga gtaaacacac 20

<210>37

<211>14

<212>DNA

<213> Artificial Synthesis

<400>37

tactcaggac tcat 14

<210>38

<211>16

<212>DNA

<213> Artificial Synthesis

<400>38

gacgatgagt cctgag 16

Claims (1)

1. The method for constructing the Pistacia chinensis Bunge microsatellite enrichment library is characterized by comprising the following steps of:

step one, Pistacia chinensis genome DNA extraction

1-1, weighing 30mg Pistacia chinensis Bunge silica gel, drying to remove petioles and veins, placing in a 2ml centrifuge tube, soaking in liquid nitrogen for 1min, and placing in a grinding instrument for full grinding;

1-2, adding 700 mu L preheated GP1 and 2 mu Lβ -mercaptoethanol into the centrifuge tube, uniformly mixing by vortex oscillation, carrying out water bath at 65 ℃ for 50min, and reversing the centrifuge tube for a plurality of times in the water bath process to uniformly mix samples;

1-3 adding 700 mu L chloroform, mixing well, centrifuging at 12,000rpm for 5 min;

1-4, transferring the upper layer into a new centrifuge tube, adding 700 mu L GP2, and uniformly mixing by vortex oscillation;

1-5 transferring the liquid into an adsorption column CB3, centrifuging at 12,000rpm for 30s, and discarding the waste liquid;

1-6 putting CB3 into the collection tube again, adding 500 mu L GD buffer solution, centrifuging at 12,000rpm for 30s, and discarding waste liquid;

1-7 adding 600 mu L of rinsing liquid into an adsorption column CB3, centrifuging at 12,000rpm for 30s, discarding the waste liquid, repeating twice, and standing at room temperature for 15 min;

1-8 transferring CB3 into a sterile centrifuge tube, suspending and dropwise adding 100 mu L ultrapure water at the middle part of an adsorption membrane, standing at room temperature for 5min to fully dissolve DNA, centrifuging at 12,000rpm for 2min, resuspending the solution obtained by centrifugation, adding the solution into an adsorption column of CB3, standing for 10min, and centrifuging at 5,000rpm for 1min to obtain a DNA stock solution;

step two, carrying out genome DNA enzyme digestion and magnetic bead enrichment on a microsatellite library

2-1 genome DNA enzyme digestion, joint connection and product pre-amplification

2-1-1 genome enzyme digestion:

performing enzyme digestion by using restriction enzyme MseI (25 mu L) containing the Pistacia chinensis genomic DNA250ng obtained in the step one, endonuclease 5U, Cutsmart 2.5 mu L and ddH₂O, a replenishing system; water bath at 37 deg.C for 1h, and water bath at 65 deg.C for 20 min;

2-1-2 enzyme digestion products are connected:

the overall ligation reaction was 30. mu. L, containing the enzyme cleavage product 15. mu. L, T4DNA ligase 4.0U, MseI linker 30pmol, ddH₂O is filled in the system and is connected for 12 hours at the temperature of 16 ℃; wherein the MseI linker sequence is specifically F: 5'-TACTCAGGACTCAT-3', as shown in SEQ ID NO: 5'-GACGATGAGTCCTGAG-3' as shown in SEQ ID NO: 38;

2-1-3 pre-amplification:

the PCR amplification system is 20 mu L, containing 10 × Buffer 2 mu L, 2.5mM dNTPs 5 mu L, 0.5U of Taq DNA polymerase and 20pmol of primer of 5 mu L-N of the enzyme digestion ligation product, wherein the sequence of the enzyme digestion ligation product is 5 '-GATGAGTCCTGAGTAAN-3';

the PCR reaction program comprises pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 60s, extension at 72 ℃ for 60s, 20 cycles, extension at 72 ℃ for 8min, and storage at 4 ℃;

hybridization of 2-2 biotin probe and pre-amplification product and magnetic bead enrichment

2-2-1 Biotin-labeled (AG)₁₅For probe and pre-amplification product hybridization:

the hybridization reaction system was 250. mu. L, in which the PCR product 30. mu. L, probe 300pmol, 20 × SSC 52.5. mu. L, 10% SDS 1.75. mu. L, ddH₂Supplementing and leveling O; after mixing uniformly, denaturation is carried out for 5 minutes at 95 ℃, and then the mixture is placed in a water bath at 48 ℃ for hybridization for 2 hours;

mixing the processed magnetic beads with the hybridization product, continuously shaking at room temperature for 50min, placing the reaction product on a magnetic frame for adsorption, and sucking out the liquid by using a pipette;

2-2-2 non-stringent washes:

washing with TEN 1000500 μ L for 3 times (8 min/time), adsorbing reaction product on magnetic rack, and sucking out liquid with liquid-transferring gun;

2-2-3 stringent washes:

washing with 0.2 × SSC +0.1% SDS mixture 500 μ L for 3 times;

2-2-4 elution:

adding 50 μ L1 × TE, water bathing at 95 deg.C for 5min, placing the reaction product on a magnetic frame for adsorption, sucking out the liquid with a pipette, and placing in another sterile 1.5m L centrifuge tube;

2-2-5 amplification and purification of enriched products:

the enriched product was amplified with MseI-N primer in a total reaction system of 20. mu. L containing 10 × Buffer 2. mu. L, 2.5mM dNTPs 2. mu. L, 20pmol primer, 0.5U Taq DNA polymerase, 5. mu. L template, ddH₂O, a replenishing system; wherein the MseI-N primer sequence is 5 '-GATGAGTCCTGAGTAAN-3';

the PCR reaction program comprises pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 60s, extension at 72 ℃ for 60s, 20 cycles, extension at 72 ℃ for 8min, and storage at 4 ℃;

2-3, detecting enriched product clone and positive clone:

2-3-1 vector ligation:

connecting the purified product obtained in the step 2-2 to a pMD19-T vector, wherein the connection reaction system comprises 4.5 mu L of DNA purified in the step 2-2, 0.5 mu L of T vector and 5 mu L of connecting liquid, the total system is 10 mu L, the reaction procedure is 16 ℃, the connection is carried out for 2h, and a PCR instrument does not use a hot cover;

conversion of 2-3-2 ligation product:

adding the connected product into 100 μ L competent cells, standing in ice for 30min, rapidly transferring into 42 deg.C water bath for 45 s, standing in ice for 1min, adding 890 μ L SOC culture medium, and performing shake culture at 37 deg.C for 60 min;

2-3-3, culturing positive clones, coating the connected transformation product in L B solid culture medium containing 100 mg/L Amp, culturing overnight at 37 ℃, picking up full and transparent positive clones, culturing in L B liquid culture medium containing Amp at 250r/min at 37 ℃ until the bacterial liquid is slightly turbid;

2-3-4 sequencing of Universal primer M13 with restriction enzyme MseI and the aforementioned Probe (AG) without Biotin labeling₁₅The formed three primer combinations carry out PCR amplification on the enrichment library, and positive clones are screened;

wherein the sequence of the restriction endonuclease MseI sequencing universal primer M13 is specifically as follows:

M13-47：5’-CGCCAGGGTTTTCCCAGTCACGAC-3’；

M13-48：5’-AGCGGATAACAATTTCACACAGGA-3’；

the primer combinations are respectively combination a: m13-47+ (AG)₁₅(ii) a Combination b: m13-48+ (AG)₁₅(ii) a And c: m13-47+ M13-48;

the PCR system is 15 mu L, which contains 10 × Buffer 1.5 mu L, 2.5mM dNTPs 2 mu L, 30pmol of upstream and downstream primers respectively, 0.25U of Taq DNA polymerase, 2 mu L of bacterial liquid and the complement of ddH2O, and the PCR reaction program comprises the steps of pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 30s, 30 cycles, extension at 72 ℃ for 8min and storage at 4 ℃;

step three, microsatellite primers

The positive clone sequencing result utilizes DNA star 8.0 to remove a vector sequence, SSR Hunter 1.3 to search a core sequence, a sequence with a longer microsatellite flanking sequence and more repeated times of a repeating unit is selected, Primer premier5.0 is utilized to design a Primer, and the principle followed when the Primer is designed mainly has the Primer length of 18-22 bp; GC content is 40-60%; the Tm value is 45-65 ℃, and the optimal Tm value is 55 ℃; the GC content difference of the positive primer and the negative primer is within 10 percent, and the Tm value difference is within 5 ℃; the product length is 200 and 400 bp; the designed primer is synthesized by Shanghai Biotechnology Limited company;

step four, detecting the effectiveness and the universality of the microsatellite primer

4-1, detection of effectiveness of primer

Performing PCR amplification on the primers obtained by the primary screening by using 24 Qiandao lake Pistacia chinensis Bunge individuals, wherein the amplification reaction system is 15 mu L and contains 10 × Buffer 1.5 mu L, 2.5mM dNTPs 1.5 mu L, 50pmol of each of upstream and downstream primers, 0.25U of Taq DNA polymerase, 1.5 mu L of a template and ddH2O, and the PCR reaction program is pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 51-59 ℃ for 30s, elongation at 72 ℃ for 30s, 30 cycles, elongation at 72 ℃ for 8min and storage at 4 ℃;

detecting the polymorphism of the amplification result by 8% non-denatured polyacrylamide gel electrophoresis and silver nitrate staining after photographing, and selecting a microsatellite primer with polymorphism;

4-2, detection of the versatility of primers

The screened primers with effective polymorphism are labeled by 6' -FAM, HEX, TAMRA and ROX, PCR amplification is carried out by using 8 individuals in one continent population and two island populations of the Qiandao lake under the same amplification condition as a PAGE gel screening system and procedure except that the annealing temperature is increased by 2 ℃, the amplification result is sequenced by using an ABI 3730X L sequencer, the sequencing result is interpreted by Genmapper4.0 and then is subjected to genetic analysis by using POPGENE 1.32 software, relevant indexes such as microsatellite allelic factor Na, observed heterozygosity Ho, expected heterozygosity He and whether Hardy-Weinberg balance is deviated or not are calculated, and the content of polymorphic information is calculated by using PIC software.

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