CN103290102B

CN103290102B - SNP (Single Nucleotide Polymorphism) classification method and application based on PCR (Polymerase Chain Reaction)

Info

Publication number: CN103290102B
Application number: CN201210053929.1A
Authority: CN
Inventors: 华玮; 黄顺谋; 王汉中; 师家勤; 刘胜毅
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2012-03-02
Filing date: 2012-03-02
Publication date: 2015-01-07
Anticipated expiration: 2032-03-02
Also published as: CN103290102A

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) classification method and application based on PCR (Polymerase Chain Reaction). The SNP classification method comprises the steps of: (1) selecting a plant sample for experiment-tissues of a plurality of strains of certain species; (2) extracting genomic DNA of the tissues; (3) purifying the genomic DNA as a template for PCR amplification; (4) selecting AT type SNP in an SNP database of the species; (5) screening out SNP of which the third base before the SNP locus is G; (6) using primer 3 software to design a primer so that the last base of a positive primer is at the SNP locus and the last base is T; (7) introducing mispairing, and changing the antepenultimate base of the positive primer into A; (8) performing PCR amplification; (9) treating the PCR products by means of modified polyacrylamide gel electrophoretic separation; and (10) observing whether amplification band exists. The invention further provides application of the SNP classification method based on PCR in pyramiding breeding; the method is easy to implement, simple, convenient, and low in cost; the detection is simple and efficient; the classification success rate of SNP is as high as 91%.

Description

A kind of SNP classifying method of PCR-based and application

Technical field

The present invention relates to molecular biology and field of bioinformatics, be specifically related to a kind of method of being carried out SNP somatotype by PCR, also relate to a kind of application of SNP classifying method at rape pyramiding breeding of PCR-based.The method is quick, and simply, cost is low, can be widely used in the field such as medical diagnosis on disease and crop breeding.

Background technology

Molecule marker is the genetic marker by between individuality based on genetic material inner nucleotide sequence variations, is the direct reflection of DNA level genetic polymorphism.With other several genetic markers---morphology marks, compared with biochemical biomarker, cytological marker, the superiority that DNA molecular marker has has: most of molecule marker is codominance, very convenient to the selection of the proterties of recessiveness; Genome mutation is extremely abundant, and the quantity of molecule marker is almost unlimited; In the different steps of biological development, the DNA of different tissues can be used for labeled analysis; Molecule marker discloses the variation from DNA; Show as neutrality, do not affect the expression of objective trait, with bad proterties without chain; Detection means is simple, rapid.Along with the development of Protocols in Molecular Biology, present DNA molecular marker technology has tens of kinds, is widely used in the aspects such as genetic breeding, genomic mapping, the assignment of genes gene mapping, the discriminating of species sibship, gene pool structure, gene clone.

The requirement of desirable molecule marker: (1) has much higher state property; (2) codominant inheritance, namely utilizes molecule marker can differentiate heterozygosis and homozygous genotype in diploid; (3) clearly allelotrope can be distinguished; (4) whole genome is spread all over; (5) except the mark of special site, require that molecule marker is uniformly distributed in whole genome; (6) select neutral (namely without polypheny); (7) detection means simply, fast (automatization as easy in experimental arrangement); (8) cost of development and use cost are as far as possible cheap; (9) in laboratory and inter-laboratory reproducibility good (being convenient to data exchange).SNP marker is the third generation DNA genetic marker that American scholar Lander E proposed in 1996.SNP meets the optimal selection of above-mentioned requirements beyond doubt.

SNP and monokaryon former times acid polymorphism mark, mainly refer to the DNA sequence polymorphism caused by the variation in genome nucleotide level, comprise the conversion of single base, transversion, and the slotting people/disappearance etc. of single base.The site of SNP is extremely abundant, almost throughout whole genome.In genome, approximately average every 1000bp just there will be a SNP according to estimates.SNP marker generally only has two kinds of allelotypes (allele) therefore is also called two equipotential mark (bi-allelic marker) in crowd or biotic population.Only need the mode of " +/-" or " all or none " when detecting like this and detect microsatellite marker without the need to picture measurement is made to the length of fragment.

The existing traditional means of means of SNP marker somatotype are as SSCP (single strand conformation polymorphism) and CAPs (Cleavage Amplification Polymorphisms) etc., also now means are faster had as MassARRAY mass spectrum and TaqMan probe method etc., to also have the detection means based on chip to develop as DNA chip technology and be employed rapidly and be tending towards ripe.Use highdensity DNA chip or micro-array can simultaneously to the somatotype of up to ten thousand SNP.Current SNP is widely used in association analysis, in gene Fine Mapping and molecular mark process.But aforesaid method all exists certain shortcoming: SSCP means can only use non denatured glue, experiment condition is harsh, and need normal temperature condition, common laboratory is difficult to reach; CAPs means experimentation is loaded down with trivial details, and an experimental arrangement needs to expend long time; MassARRAY mass spectrum and TaqMan probe method and other chip method appropriate litigation fees high, common laboratory is difficult to bear.

The present invention is based on above consideration, realize one both cheap, simply, the SNP classifying method that efficiency is high again, during by design primer, introduce specific mispairing type and mismatch site at 3 ' end of forward primer, make primer pair very high containing the efficiency of pcr amplification in the genotypic material of a certain SNP, and the efficiency of pcr amplification is very low in the material of another kind of SNP site, thus reach the object of SNP somatotype.The present invention is under the experimental procedure condition the same with SSR molecular marker, greatly improves the ratio of primer polymorphism, expands the region of covering gene group greatly.SSR molecular marker is two storeroom polymorphism ratios generally about 30%, and molecule marking method of the present invention can reach more than 90% two storeroom polymorphism ratios.Under the prerequisite of experimental cost the same as SSR molecular marker, molecule marking method of the present invention has higher efficiency.

Summary of the invention

The object of the invention is the SNP classifying method that there are provided a kind of PCR-based, during by design primer, specific mispairing type and mismatch site is introduced at 3 ' end of forward primer, make primer pair very high containing the efficiency of pcr amplification in the genotypic material of a certain SNP, and the efficiency of pcr amplification is very low in the material of another kind of SNP site, thus reach the object of SNP somatotype.Compared with many existing methods, its major advantage is that simply, cost is low, can be widely used in the field such as medical diagnosis on disease and crop breeding fast.

Another object of the present invention is the application at rape pyramiding breeding of the SNP classifying method that there are provided a kind of PCR-based, molecule marker conventional is at present SSR, this molecule marker distributes less in genome, and being mainly positioned at non-coding region, this characteristic hinders the exploitation of close linkage assisted Selection mark.And the present invention uses SNP marker, SNP marker has a very wide distribution at genome, and is extensively present in gene coding region.Find in human genome research, only about half of non-synonym base mutation is source of causing a disease.And SSR is only closely linked molecule marker, cause the possibility of pathology little.In pyramiding breeding process, what be in fact polymerized is superior allelic, if assisted Selection mark SSR, there is the possibility that target superior allelic is lost in assisted Selection process, because the restriction of SSR self character, can not be divided into target superior allelic all the time from.And SNP marker can be positioned at coding region, and may be the excellent source of superior allelic, and the possibility that SSR becomes the excellent source of superior allelic be very little.So application the inventive method, can use on the basis of same cost with SSR, reach better effect.

To achieve these goals, the present invention is by the following technical solutions:

A SNP classifying method for PCR-based, the steps include:

1, to select in two brassica napus two No. 11 and 73290, get 5 grams of leaf samples;

2, the genomic dna of tissue is extracted after leaf sample liquid nitrogen freezing grinding step 1 obtained; The method wherein extracting DNA in step is CTAB small-sample method.

3, using the template as pcr amplification after Genomic DNA Purification; Wherein in step purify DNA be the test kit of AXYGEN company, carry out according to instructions to the user.

4, in two develop between No. 11 and 73290 liang of strains snp database in have 4 type SNP:A/G (C/T), A/C (G/T), A/T, G/C.Select the SNP of A/T type for next step.The SNP selected in this step be by rape two strains (in two No. 11 and 73290) solexa resurvey the SNP that sequence 5X data analysis obtains, SNP by the degree of depth of reads support minimum be 4 layers.

5, in the result of step 4, to filter out before SNP site the SNP that the 3rd base is G;

6, design primer with primer3 software (http://primer3.sourceforge.net/releases.php), make last base of forward primer be positioned at SNP site, and last base is T; Wherein in step, during primer3 software design primer, Tm value is minimum is 62 DEG C, and be 68 DEG C to the maximum, Optimal Temperature 65 DEG C, expanding fragment length is between 300 to 500bp.

7, introduce mispairing, third from the bottom base of forward primer is become A; Primer is PrimerID01-23.

8, pcr amplification is carried out according to the reaction conditions of design;

9, PCR primer step 8 obtained is electrophoresis under specific voltage conditions and specific denaturing polyacrylamide gel concentration; Wherein suitable in step deposition condition is: the electrophoresis system using the DYCZ-20C type of Liuyi Instruments Plant, Beijing, and deposition condition is electrophoresis 80 minutes under 1800V voltage.

Described specific voltage conditions is that the heat produced in electrophoresis process can be fully lost, and unlikely electrophoresis temperature is too high because under this voltage conditions.Under described specific denaturing polyacrylamide gel concentration, electrophoresis is because under this concentration, and the mesh that gel produces can just reach the object that DNA fragmentation is separated.

10, observe the presence or absence of amplified band after gel silver dye step 9 obtained, have the brightness of band very high, the brightness without band is very low even not to be had, and reaches the object of somatotype two kinds of SNP types.Wherein in step, the silver nitrate concentration of silver dye is 10 μ g/ml.

Present method is verified equally in sesame and soybean, all reaches the polymorphism ratio of more than 90%.

The SNP classifying method of PCR-based, in an application for rape pyramiding breeding, the steps include:

1, find the particular line improveing some defect proterties of backbone parent, hybridize with backbone parent and prepare mapping population.

2, sequence of resurveying particular line and backbone parent, search the SNP corresponding to the present invention, do genotype identification at mapping population, builds genetic map.

3, mapping population is done the field test of multiple spot, obtain phenotypic data, then with genetic map binding analysis Mapping of QTL, find the superior allelic type of closely linked SNP marker and correspondence.

4, in the strain of mapping population, select the strain containing many superior allelic type as far as possible, then backcross with backbone parent, until remain all superior allelic types, till reaching the object of improvement backbone parent.

The present invention compared with prior art, has the following advantages and effect:

1, easy to implement the method, easy and simple to handle, cost is low.Current SNP typing method, as TaqMan probe technology, synthesizes a probe and needs about 1000 yuans, and we this invention only need to synthesize one to one as primer about 60 yuans;

2, detect easy.If CAPS is when somatotype SNP, need to do DNA purifying and enzyme is cut, experimentation is loaded down with trivial details, and the present invention only needs to run PCR and adds gel detection, and experimentation is simple;

3, somatotype equipment is simple.Need fluoroscopic examination as during TaqMan probe method somatotype, apparatus expensive about needs 700,000 yuans.And the present invention only needs general electrophoresis equipment, only about need 4000 yuans;

4, efficiency is high.The somatotype success ratio of SNP of the present invention reaches 91%, and this is that SSR (simple repeated sequence) molecule marker institute can not be than;

5, flux is controlled.SNP classifying method at present based on TaqMan probe and chip technology is only applicable to the SNP somatotype that sample size is large or SNP site amount is many, and to general QTL (quantitative trait locus) Position Research, SNP classifying method of the present invention is the most applicable, can meet the needs of common laboratory.

Accompanying drawing explanation

Fig. 1, be a kind of design of primers schematic diagram.

Last base of forward primer is positioned at SNP site, and be base T; 3 ' holds antepenultimate base to be originally G, becomes A.

Fig. 2, be a kind of 23 SNP genotyping result schematic diagram.

A: in two No. 11; B:73290; No. 1-23:1-No. 23 SNP; The amplified band luminance difference of No. 03 SNP two materials is not obvious, and No. 14 SNP all do not increase out at two materials, and remaining 21 SNP site all can detect polymorphism well.

Fig. 3, be a kind of genetic map schematic diagram.

Ns162 and ns163 is the molecule marker of method of the present invention exploitation, is the closely linked molecule marker of rape proterties resistant to lodging.Other are labeled as the skeleton mark of QTL designation of chromosome resistant to lodging and SNP marker not closely linked with QTL resistant to lodging, and asterisk represents QTL region.

Embodiment

Usual conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or people (the Blackwell Science Press such as Draper, 1988) condition described in, or according to the condition that agents useful for same manufacturer advises.Agents useful for same is as without special instruction, all available in biochemical reagents shop, and software used is freeware.

Embodiment 1:

A SNP classifying method for PCR-based, the steps include:

1. utilize CTAB method to extract blade STb gene:

A., after in appropriate swede type rape pair No. 11 and 73290 leaf samples being taken out from Ultralow Temperature Freezer (-70 DEG C), put into frappe mortar immediately, add liquid nitrogen grinding powdering; In quick loading 50ml centrifuge tube, be added in extracting solution (0.2M Tris-Cl, 0.25NaCl preheated in the water-bath of 60 DEG C, 25mM EDTA, 0.5% (mass ratio) SDS, pH 7.5), mix, put into the water-bath water-bath 40min of 60 DEG C;

B. take out centrifuge tube, add equal-volume chloroform: primary isoamyl alcohol (24: 1, V/V), slowly turn upside down centrifuge tube 30-50 time, makes abundant mixing, centrifugal 10 minutes of 1300g;

C. get supernatant in another centrifuge tube, add equal-volume chloroform: primary isoamyl alcohol (24: 1, V/V), extracting once again.Get supernatant, add 0.6 times of volumes ice cold primary isoamyl alcohol, slowly put upside down centrifuge tube, till having flocks to assemble.Static 30min, chooses precipitation, and 70% (volume ratio) alcohol is washed 2-3 time, and dehydrated alcohol is washed once, adds sterilized water 65 DEG C of 20min and dissolve after drying;

D. equal-volume chloroform is again added: primary isoamyl alcohol (24: 1, V/V), extracting again.Get supernatant, add 0.1 times of NaAc (3mol/L, pH5.2), slowly add the ice dehydrated alcohol of 2 times of volumes after mixing, slowly rotate centrifuge tube until flocks occurs after static 5min, choose precipitation and proceed in 1.5ml centrifuge tube, 70% (volume ratio) alcohol is washed 2-3 time, dehydrated alcohol is washed once, adds sterilized water and dissolves, save backup in-20 DEG C of refrigerators after drying.

The screening in 2.snp site:

In two No. 11 and 73290 each 1ug of DNA send Hua Da gene studies institute by solexa order-checking acquisition 5X sequence data.The genome sequence (chiff-401and 02-212) of Chinese cabbage and wild cabbage is merged as the genomic reference sequences of swede type rape, then use 2bwt-builder software ( http://soap.genomics.org.cn/soapaligner.html) with reference to Format Series Lines, then the sequence SOAP software (http://soap.genomics.org.cn/soapaligner.html) two material order-checkings obtained and the reference sequences comparison (alignment parameters-v5) after formaing, then screening obtains the comparison result of Matching Uninqueness position, analyze with soapsnp (http://soap.genomics.org.cn/soapsnp.html) and obtain a large amount of SNP, two material reads support that the degree of depth all needs to reach more than 4X, real SNP between two No. 11 and 73290 in just calculating, extracting in the SNP information obtained is A/T type again, 3 ' end left several three be the SNP site of G, for next step design of primers.This have selected 23 SNP site altogether, and its site information is for shown in SNP01-23.

3. design of primers:

First determine forward primer, the optimal T m value of primer is set to 65 DEG C, minimum 62 DEG C, the highest 68 DEG C.Last base of forward primer is positioned at SNP site, and be base T, and antepenultimate base is originally G, becomes A, make primer to 5 ' end extend (Fig. 1), with oligotm software ( http:// primer3.sourceforge.net/releases.php) the Tm value of forward primer is detected until Tm value reaches optimum.Forward primer determines reverse primer after determining again, after utilizing the fixing forward primer position of primer3 software (http://primer3.sourceforge.net/releases.php), search for reverse primer again, make the Tm value of reverse primer consistent with forward primer, and the length of amplified production is between 300-500bp.According to selected SNP site information, 23 pairs of primers of design are for shown in PrimerID01-23.

Primer with in two No. 11 DNA profilings in conjunction with time, only there is artificial a pair base mismatch introduced between primer and template, to A-C, does not affect the efficiency of pcr amplification.And with 73290 DNA profiling in conjunction with time, owing to there are two base mismatch, the T-T mispairing of SNP site and the A-C mispairing of the 3rd of 3 ' end, cause the efficiency of pcr amplification extremely low, gel almost can't detect amplified production.

4.PCR amplification and gel somatotype:

(1) PCR system and program:

PCR response procedures:

(2) gel electrophoresis:

Preparation of reagents:

6% (mass ratio) PAGE glue: 6% (mass ratio) acrylamide, 7mol/L urea, 0.5 × tbe buffer liquid, adds 300ul 10% (mass ratio) NH before encapsulating ₄s ₂o ₄, 30ulTEMED; 10 × TBE:Tris-base 108g, boric acid 55g, 0.5MEDTA (pH8.0) 40ml, being settled to dilution when 1000ml. uses is 0.5 × TBE working fluid.

Prepared by PAGE glue:

Offset plate 10% (mass ratio) NaOH solution soaks 24 hours, cleans, airing.The anti-silanizing agent of long offset plate medicated napkin uniform application (AMMMRESCO), 1ml silanizing agent (0.5% (volume ratio) Glacial acetic acid smeared by short offset plate, 1.5ml95% (volume ratio) ethanol, 1-2ul silanizing agent), place after 5 minutes, shampoo gently to remove unnecessary anti-silanizing agent and silanizing agent with the ethanol of 95% (volume ratio).Glass is installed, and separates with edge strip, be carefully inserted in glue folder (GIBCO/BRL).After ready on short offset plate with syringe by 50ml (long offset plate 80ml) sex change glue mixed solution, slowly inject, so as not to produce bubble.Insert comb, and clip with clip, condense and get final product electrophoresis after 2 hours.

Electrophoresis:

From glue folder, take out offset plate, clean glass outer side, be fixed on electrophoresis chamber, upper and lower groove respectively adds 500ml 0.5 × tbe buffer liquid, rinses loading wells immediately after being extracted by comb, and switch on power 1500 volts of constant voltage preheating 30min.Isopyknic sample-loading buffer (98% (volume ratio) deionized formamide is added in PCR primer, 10mmol/L EDTA, the blue or green FF of 0.005% (mass ratio) dimethylbenzene, 0.005% (mass ratio) tetrabromophenol sulfonphthalein), 95 DEG C of sex change 5 minutes, ice bath cools, loading 5ul, 1800 volts of constant voltage electrophoresis 80min.Electrophoresis is stopped when the blue or green FF of dimethylbenzene arrives 2/3 offset plate.

Dyeing:

Argentation is adopted to dye: it is colourless that 10% (volume ratio) Glacial acetic acid is fixed to offset plate, ddH ₂o rinsing twice, each 2-3 minute.Then dye 0.1AgNO ₃, 0.56% formaldehyde (volume ratio) 30 minutes.Take out offset plate, at ddH ₂o rinsing 10s, then at developing solution (3% (mass ratio) Na of precooling (4 DEG C) ₂cO ₃, 0.56 (volume ratio) %HCHO, 2mg/LNa ₂s ₂o ₃.5H ₂o) shake to band high-visible in gently, then pour into and stop, in liquid (10% (volume ratio) glacial acetic acid), to stop development.With distilled water rinsing 3 minutes, natural airing under room temperature (20-25 DEG C), preservation of taking pictures.

5. interpretation of result:

As shown in Figure 2, from left to right carry out the somatotype of 23 SNP site altogether, a in being two No. 11, b is 73290, can find out in 23 sites, there are 21 SNP site polymorphism (being embodied by the presence or absence of amplified band) all can be detected well, obtain genotyping result more clearly.Only the amplified band luminance difference of No. 03 SNP two materials is not obvious, and No. 14 SNP all do not increase out at two materials.Somatotype success ratio reaches 91%.

Embodiment 2:

Farm crop conventional molecular breeding method is polymerized multiple good character and is often used SSR marker, and this to be marked at genomic density lower, is difficult to find the closely linked mark of functional gene.Apply indicia means of the present invention, the inaccessiable linkage degree of SSR marker can be reached, greatly improve efficiency.Two No. 11 (state examines oil 2008030) and 73290 materials in current material, wherein, two No. 11 have cracking resistance angle, resistant to lodging, anti-sclerotium disease, angle fruit is long, and thousand seed weight is large waits good character, and 73290 to have branch amount many, complete stool angle fruit number waits good character.Present target be by good character transformations many for many for the branch amount of 73290 materials and complete stool angle fruit number in two No. 11 materials, reach the object of in improvement two No. 11.

1, F2 is for the structure of mapping population.With in two No. 11 be maternal, 73290 is male parent, artificial hybridization acquisition F1 generation, then by F1 generation selfing, obtains a lot of F2 generation.Get F2 and extract DNA for single-strain blade, as the DNA sample of F2 mapping population, after giving over to, do genotype identification.Then by F2 for the selfing of individual plant whole branches, point individual plant results selfed seed, as the strain of F2 for mapping population, for phenotypic evaluation after giving over to.

2, SNP exploitation.In sequence of resurveying two No. 11 and 73290, by the DNA extraction of two materials out after, be purified into each 1ug and be supplied to Hua Da gene for order-checking, check order and read long 100bp, order-checking amount 5G.Sequencing result is with the comparison of SOAP software (parameter-v5), the reads extracting Matching Uninqueness position from comparison result out identifies for SNP, SNP qualification is done with SOAPsnp, the SNP of A/T type is extracted again out in SNP qualification result, and 3 ' holds antepenulatimate to be the SNP of bases G, SNP site information is for shown in SNP01-23.

3, map construction.According to the primer design method design primer in embodiment 1, primer sequence is for shown in PrimerID01-23.Then genotype identification is done, the 4th step in concrete steps reference example 1 for the DNA sample of mapping population to the F2 that the present embodiment the 1st step obtains.Obtain mapping population genotype data after, with joinmap3 ( http:// www.kyazma.nl/index.php/mc.JoinMap/sc.Updates/) genetic map (Fig. 3) is built.

4, phenotypic evaluation.The first step of the present embodiment is obtained F2 does multiple spot field test for the strain of mapping population, investigate proterties, obtain resistant to lodging, cracking resistance angle, anti-sclerotium disease, complete stool angle fruit number, Pod length, angle fruit grain number, the phenotypic evaluation data (table 1) of the proterties such as thousand seed weight.

5, the proterties phenotypic data resistant to lodging of table 1. mapping population

6, QTL location.3rd step of the present embodiment and the result of the 4th step are input to WinQTLCart2.5 software (http://statgen.ncsu.edu/qtlcart/WQTLCart.htm) locate for QTL, permutation test is set to 1000 times, carry out QTL scanning, obtain the QTL data of multiple proterties.

7, strain is selected and is backcrossed.From F2 for selecting containing the genotypic strain of excellent QTL (the QTL allelotype to positive result proterties resistant to lodging) in the strain of mapping population, again with in two No. 11 backcross, until both obtained all 73290 superior allelic types, two No. 11 original good characters in remaining again thus till reaching the object of in improvement two No. 11.

8, the SNP marker assisted Selection having utilized the present invention to obtain at present obtains multiple improvement strain, for product ratio.

SNP site information is as follows:

SNP01

AAAGAGAGGTGACGATGAAACTTGAGACCCAGTAGATTCGTAACGTTAACGGATGGTTGG[A/T]TGATGCGAATCTTTTCTGGTGGAGAACCAAAACGGCGATGACGGCGTAAGTAACGGCGTG

SNP02

TTCTGTGACATTCTTCTGTAAAGCCTCTTCAACATTTGGTGTTGTGTTCTCTTGTTGGGA[A/T]TGATCTGTGTTGGGCTGCAAAGGAGGAGGCTGGGATGGTAAGTTCTCTGTCTGAGATGCT

SNP03

TGAGGATGAGATGGTGATTCACGTAAACAAATCAGCAAATCCTTCATCTGAGGACTATGG[A/T]ATACCATCGCAGCTTGTGCAGAAATATGTTAAAGGTACATTTATCTTCACTTGAGTCCTT

SNP04

ACCAGCTTAGAAACCAGCTGAGATAGATCAGCATAGAGGAGGCAAGCCAAAGACTGAGGA[A/T]CACATGGTGCATTGTAGTAGTTACATACCTTACGAACAACACCCCCCCCCCCCCCCCCCC

SNP05

GATGTAATGGTTATGGGCACATAAAATTAGAGTGTGTGAATACAAAGAAGATGAAGATGA[A/T]GAGAAGTTGCAGTGATAAGAAGCCAGAGAGAAAAGAAGATGCTCTAAATTTTGTGGCTCT

SNP06

GATCCTAAAGATTCACAATACTCTAAACCCAGCAGATATGTTGACCAAGTGTTTACCTGG[A/T]AGCGCGTTCGAGAAGTGCCTCGTAACGCCGAGGGTCACCGTCTGATCATGTGTCGAAGTA

SNP07

CTTTCTTCCTTACGACATACGCCTCAAGGAGCCTCACGCAGCAGTTTCAGGGATAGTAGG[A/T]AGCACCTTGGCAGCTTGGATCACAGGCCGAGACACGAAGAGGAGGAGTCCGATAACTGCA

SNP08

CATCGCCACTGAGATATGCGCAGCGCTTGTCTTTCTTCACTCCAATAAATCTCATAGCGT[A/T]GTCCACGGTGATTTGAAGGCGGCGAGTGTTCTTCTCGATGCTAATCTCGTTAGCAAGTTA

SNP09

GAGCACTCTCCAGGAAACGCCCTGCCTTTAGAGAGAAGAAGATTCAAGAGGAGTCTCGGA[A/T]CGACACAGACAGAACAAGAACTGAGGAGGCAAAAGACACGAATCTAAACAGTCGCAGACA

SNP10

GAGGCTAAATATTCAAGAAAAAAAGGAAGCTTTCATTCAATAAAAGCTAATAGGCAACGT[A/T]GTATAGGTCCATCAAAAGCTAAAACAGAGTGTAGAGAGCCTAACACAATCATACAGCAAA

SNP11

ATGCATAGCCTCTTCCTCTCGCTCACAGCAGCTCTAGGACTCGGTGGCCTCCCCCTTTGT[A/T]GCATGCTTGCATACGTACTCGGATATGGAAAACCTAGAATTGCACCCGCAGTTGGAGAGA

SNP12

GTATTACCGCAGACACAAAGTCTGTGAGGTTCATGCAAAGGCCTCTTCTGCAACTGTTGC[A/T]GGAGTCAAGCAACGTTTTTGTCAACAATGCAGCAGGTAACCATTTTTCCCTTCAAATATA

SNP13

AAGAGATTCATGCTTAGTCGACACAGCGAGAAACTTGCAATGGCCTTTGGTCTCATCAGC[A/T]CAAACAAAGGCACAAGAATAAGAATAGTAAAAAACCTGAGAGTATGTTCTGATTGTCATT

SNP14

ATTATTGGCTTCAGGTGCGAGGGGAAAGGTGGCTGCACCATGGAGGTCTGATGTAGATGG[A/T]TGGGATGGTCCTAGCTATGCTTCTTCGAGAAACGCTCAGACCAGTTCGAGAAAGACACTT

SNP15

AGTGGTTAAGGAATCTCTGGAAGAAGTTGAAGTTGAGTACTTAATGTTAGCAGAGGAAGC[A/T]TTAGGCAATGACACTTATGATGAGGATGTATGGATGATATACCCCGATGGTACAACGAAT

SNP16

ATAGAATATCAGAAACACTAGAGGCAATAGTGTCAGCTTTGGACTTGGTTTTGTCTTGGC[A/T]ACATTTTCATCGTTTGCGGTTGTCTCTTCACCCATCAGACAAGTACCACCGGCAGATTTG

SNP17

AGGACGATGTCACTACTCCGAGCCTGGATTGAGATCATTCAGGCGGCGCAAGCCTATTGA[A/T]GACGGCAAGACCTCTTGAGAATGCTCTGAGGGATAAATTTCGATCAGCTCTAACTAGTTT

SNP18

AGTCCTTTACAAACCAGAGATCTAGCAGAGATCATACTTCTCCAGCCATATGACGGGAGT[A/T]GGATCGGATCGGTTCAAGGGGTGAAGCATTCCTATAGTACCGTCCTTTAAAGACTCTAGA

VSNP19

CTATGATGCAAGGAACGAGTTACCTGGGTTCGATCTTCACTTGGGACCAAACAAATGGGA[A/T]AGTGTTAAACTGGAGTCTTCTGAGGGAACAGTTTCCAAAGAGATTATATACTCTGTCTTG

SNP20

ACAGTACGGGAAACACTGGTGGTGGCTCTCTTTCTTCTCCGTCTACGTTTCTCAGCAGGT[A/T]AAGATCCACCAACTGTTCGATGATTTGCCTCAACGAGTAGATTTTCATTTGGTTCTTTTG

SNP21

TGGTATCTTCTTTTTTTTTTTTTTTTTTTTTGTTTACAGGAGGTTCAAAGGTGAACCCGT[A/T]ATCCGCACGTGGTTTCCGTTTTGCCCAAAGGCCCGTAATCTGCACGTTGTTTCCGATTGC

SNP22

TAAGCTCACCTGCAGGCAATTTTATTTTTTGATTGATGTTAGTGATTTCAACTGGAATGA[A/T]ACATCTACATGTCTCCATATTTATATGGAGAAAGGAGGTAAACCACAGTGAAGAGAAATA

SNP23

AATCCAAACATCCTTAAAAACCTCATTTTTTATAAAAGCATGGTTCAAACACAAGGCCGG[A/T]AAAAGGACTTACTCGGCCAGTCCAATTACATAAAAAAACGGCAATACTGGGCCAGCACAC

The primer information of somatotype 23 SNP is as follows:

PrimerID	Forward primer sequence	Reverse primer sequences
			Primer01	CCAGTAGATTCGTAACGTTAACGGATGGTTAGT	GTCGATCTACTCTTCCTCATCATCCTAACCTTCTT
Primer02	AACATTTGGTGTTGTGTTCTCTTGTTGGAAT	ATGCATCTATGAATGAAGGAGATGAAACTAATGATG
			Primer03	ACAAATCAGCAAATCCTTCATCTGAGGACTATAGT	CGGACTTTGGTGGCCACTGGAAT
Primer04	AGAGGAGGCAAGCCAAAGACTGAGAAT	CAGCTATTTCTCTAGCGTGAGATGTTCCTGAGTAT
			Primer05	ATTAGAGTGTGTGAATACAAAGAAGATGAAGATAAT	TCAATTTGTTCCATCTCTAGGATGTTGACTTGA
Primer06	CCAGCAGATATGTTGACCAAGTGTTTACCTAGT	TAAGTTTTCTTCCTTACACAACTCGAATCTCCCTT
			Primer07	CTCACGCAGCAGTTTCAGGGATAGTAAGT	TCATCACACTACTTTGGAACAAGAAGCTTAAGTACC
Primer08	CTTGTCTTTCTTCACTCCAATAAATCTCATAGCATT	CCGGACGATTCTCACTGACAGTCTCAC
			Primer09	CTTTAGAGAGAAGAAGATTCAAGAGGAGTCTCGAAT	GAAGACAACAAAGCTTCGACCTTTGCAAT
Primer10	GAAGCTTTCATTCAATAAAAGCTAATAGGCAACATT	TTTTGTTTACCGTGGTGTTAATAGGACTCTGTTTTT
			Primer11	GACTCGGTGGCCTCCCCCTTTATT	GTTGAGGAAGAATGGCGTTCTCACCAT
Primer12	GCAAAGGCCTCTTCTGCAACTGTTACT	GGTTGGGTGAGTTGATATTGTTTTATCTGATCTGT
			Primer13	GCAATGGCCTTTGGTCTCATCAACT	TGCTGAGCTTTGGGGTTCTGATCCT
Primer14	GCTGCACCATGGAGGTCTGATGTAGATAGT	GCTCCAACAAGTCCAGACAGGGTCC
			Primer15	GTTGAAGTTGAGTACTTAATGTTAGCAGAGGAAACT	CTGCTCCAGTCCGGTCTAGAAAAGTGC
Primer16	TCAGCTTTGGACTTGGTTTTGTCTTGACT	AACGTCACATCCAATCTTGAATCTGAGAAGTAAA
			Primer17	TCAGGCGGCGCAAGCCTATTAAT	CCAGTTATGTCCCTCTCGATTGACAGACA
Primer18	TCATACTTCTCCAGCCATATGACGGGAATT	AGTCGGTGGTTACGACTCTGCCAAATC
			Primer19	TCTTCACTTGGGACCAAACAAATGGAAT	GAAACTTACCGCTACTGTTGCGTGAGGA
Primer20	TCTTTCTTCTCCGTCTACGTTTCTCAGCAGATT	GATGCCTCGAGTAATACCACAAACCCG
			Primer21	TGTTTACAGGAGGTTCAAAGGTGAACCCATT	TTATGTTATCTCCATTGCTAGTGTTCATGTGATCAG
Primer22	TTTTTGATTGATGTTAGTGATTTCAACTGGAATAAT	ATGCGATAGAAATGGGTGTTTTCTAAAGATGTG
			Primer23	AAAGCATGGTTCAAACACAAGGCCAGT	AATTTCTGAGGTTGAGGAAGAGATTTGGGAG

Claims

1. a SNP classifying method for PCR-based, the steps include:

A, the tissue selecting for the plant sample of testing, plant sample is the leaf sample of two swede type rape strains: in two No. 11 and 73290;

B, the rear genomic dna extracting tissue of plant sample liquid nitrogen freezing grinding that steps A is obtained;

C, using the template as pcr amplification after Genomic DNA Purification;

D, select the SNP of A/T type at the snp database of swede type rape, the SNP selected in this step is by swede type rape two strains: in two No. 11 and 73290, solexa resurvey the SNP that sequence 5X data analysis obtains, SNP by reads support the degree of depth minimum be 4X;

E, in the result of step D, to filter out before SNP site the SNP that the 3rd base is G;

F, use primer3 software design primer, make last base of forward primer be positioned at SNP site, last base is T; In this step, during primer3 software design primer, Tm value is minimum is 62 DEG C, and be 68 DEG C to the maximum, temperature 65 DEG C, expanding fragment length is between 300 to 500bp;

G, introducing mispairing, become A by third from the bottom base of forward primer; Described primer is for shown in PrimerID01-23, and the corresponding sequence including SNP site is for shown in SNP01-SNP23;

H, according to design reaction conditions carry out pcr amplification;

J, PCR primer electrophoresis under specific voltage conditions and specific non-denaturing polyacrylamide gel concentration that step H is obtained, deposition condition is electrophoresis 80 minutes under 1800V voltage;

K, the rear presence or absence of observing amplified band of gel silver dye that step J is obtained, the silver nitrate concentration of silver dye is 10 μ g/ml;