CN102533727A

CN102533727A - Preparation method of developing Phaseolus vulgaris SSR primers by magnetic bead enrichment

Info

Publication number: CN102533727A
Application number: CN2011104370945A
Authority: CN
Inventors: 汪爱华; 周国林; 林处发; 丁毅; 胡侦华; 黄兴学; 邓耀华; 王斌才; 姚芳
Original assignee: Wuhan vegetable research institute
Current assignee: Wuhan vegetable research institute
Priority date: 2011-12-23
Filing date: 2011-12-23
Publication date: 2012-07-04
Anticipated expiration: 2031-12-23
Also published as: CN102533727B

Abstract

The invention discloses a preparation method of developing Phaseolus vulgaris SSR primers by magnetic bead enrichment. The method comprises the following steps of: (1) digesting Phaseolus vulgaris genome DNA with Msel, and establishing a library by AFLP; (2) hybridizing the genome PCR library with a biotin-labeled repetitive sequence probe; (3) adding the hybridization mixed liquid into streptavidin-coated magnetic beads; (4) amplifying micro-satellite DNA fragments by PCR using eluent-purified micro-satellite DNA as DNA template; (5) linking the purified PCR amplification product on a T vector for cloning to obtain a DNA sequence with inserted fragments; and (6) repeating on the two sides of the core area to design primers, and screening primers to obtain effective microsatellite loci. The method is simple and feasible, increases efficiency of the primers, screens 20 pairs of effective primers from 48 pairs of primers, and provides guarantee for large-batch development of SSR primers.

Description

A kind of preparation method of enrichment with magnetic bead method exploitation Kidney bean SSR primer

Technical field

The invention belongs to bioengineering field, relate to a kind of preparation method of enrichment with magnetic bead method exploitation Kidney bean SSR primer, this method is applicable to all Kidney bean kinds, strain or germ plasm resource material.

Background technology

Though the SSR mark is effective, safety is high, and its key point is the exploitation of SSR primer again.Only after the SSR primer of some amount is arranged, just possibly carry out the analysis of SSR mark to a certain species.Particularly for the living species that did not check order, for its DNA base sequence know little about it or the species known nothing can only utilize some special methods obtain this Tumor-necrosis factor glycoproteins with and the flanking sequence on both sides design the SSR primer.The source of SSR primer is summarized and is got up to have following four kinds of approach: 1. closes document at present; 2. the primer of sibling species (for example belonging to together not of the same race); 3. database search method: in public databases such as GenBank, search the SSR sequence, according to flanking sequence design primer; 4. the own SSR site both wings sequences Design primer that from the research object genomic library, screens.To those great majority order-checkings the 4th kind of methods of species seldom almost is unique channel, also is the most basic, effective means.The method that obtains the SSR site from genomic library has comprised classical way, concentration method and omission sieve storehouse method at present again.

Set up the genomic library of the species of studying, hybridize with it with the probe that contains SSR then, screening positive clone, order-checking is according to the dna sequence dna design primer of SSR both sides.This traditional method can be divided into two kinds again: the one, and a kind of is the big fragment gene group library of inserting of screening, and demonstrating with probe has the clone of strong hybridization signal to be further purified, and is divided into subclone, and another screening by hybridization contains the clone of Tumor-necrosis factor glycoproteins then.Another kind is that genomic dna is become the small segment of 200-1500bp with digestion with restriction enzyme, also can use the ultrasonication genomic dna, and the fragment that obtains is littler relatively, and 200-600bp mends its flat with the big fragment of Klenow of dna polymerase i again.Make up and screen little insertion fragment gene group library.Two kinds of methods respectively have shortcoming, first kind to big subclone be difficult to the order-checking, second kind of yield that obtains the SSR positive colony is very low, has only 2%-3%.The efficient of this traditional classical method exploitation primer is very low, but simple to operate, is prone to grasp, and in existing acquired SSR mark, the classical way that utilizes more than 3/4ths is developed.

Concentration method has improved SSR clone's yield through setting up and the screening enriched microsatellite library.It is divided into again and utilizes the PCR primer contain SSR to carry out PCR enrichment (being the enrichment of primer method) and hybridize two kinds of methods of enrichment (being the hybrid method enrichment) with the probe that contains SSR.

The little satellite of primer method enrichment is meant that generally use repeats the complementary oligonucleotide as primer with the little satellite of purpose, is reflected at before the library construction or makes up back enrichment microsatellite locus through PCR.Elaine etc. are material with the genomic dna of dog; Adopt classical way to obtain the segmental primary gene group of a little insertion library earlier; Nucleotide (CA) n with 5 ' phosphorylation is that primer carries out a step PCR then, is transformed into bacterium, obtains one and contains (CA) n enriched library.Film is changeed in picking mono-clonal cultivation back at random, hybridization, and screening once more, the result who obtains is: 40%-50% is a positive colony, has improved 50 times (Elaine et al., 1992) than traditional method efficient before.

Use more primer development strategy to be based on the enriching method of selective cross at present, representative is two kinds of hybridizing methods, is divided into nylon embrane method and enrichment with magnetic bead method according to employed medium difference.The nylon embrane method is come the little satellite fragment in the enrichment genome through the many little satellite probe that is adsorbed on the nylon membrane; The elution of bound fragment; Carry out pcr amplification; The connection carrier transformed into escherichia coli; The positive colony order-checking.Use the nylon embrane method, 6iovannim repeats probe with 11 little satellites and from Saccharum spp, screens microsatellite locus, and bioaccumulation efficiency has improved 40%.The ultimate principle of enrichment with magnetic bead method is at first to hybridize with biotin labeling Tumor-necrosis factor glycoproteins specific probe and with the genomic dna endonuclease bamhi; Utilize vitamin H and the strong characteristic of streptavidin affinity again, carry out the enrichment that the Tumor-necrosis factor glycoproteins target fragment is accomplished in magnetic force absorption with the magnetic bead that encapsulates streptavidin.Through the cloning and sequencing amount being declined to a great extent behind the enrichment with magnetic bead and the segmental efficient that obtains to have Tumor-necrosis factor glycoproteins being increased substantially, therefore, become extremely effective means and being used widely rapidly of SSR primer development.Utilize the report of enrichment with magnetic bead method developing SSR primer to have much at present.

Records Original the like Li Masson nuclear genomic DNA Sau3A? I digested fragment was recovered 300-1000bp, connectors, PCR and labeled with biotin after microsatellite probe (AC) 15, (AG) 15 hybridization; using magnetic Beads enriched microsatellite sequences containing DNA fragments.The sequence that obtains through behind the pcr amplification, is connected the pGEM-T carrier, be transformed into the competence intestinal bacteria, obtain the microsatellite sequence library.And then directly to the library by PCR amplified to obtain 58 positive clones were sequenced, won 33 microsatellite sequences, and finally successfully designed Masson microsatellite primers 19 pairs (Li Records Original etc., 2007).

Sun Xiaowen etc. cut grass carp (Ctenopharyngodon idella) genomic dna through the Sau3AI enzyme, reclaim purifying 400-900bp fragment, connect joint, make up in " genome PCR library ".Do probe and its hybridization with biotin labeled (CA) 15, hybridization complex is attached on the magnetic bead that is coated with Streptavidin, and through a series of washing process, enrichment contains the fragment of little satellite.Pcr amplification amplifies little satellite fragment, clones and checks order, according to the conserved sequence design primer at little satellite two ends; And carry out the secondary screening by hybridization through isotope-labeled probe (CA) 15, and obtaining 132 of positive colonies, resulting positive colony is through order-checking; 86.36% contains microsatellite sequence; Obtain 130 microsatellite DNA sequences altogether, design SSR primer 83 is to (Sun Xiaowen etc., 2005).

Need only the dna fragmentation that just can obtain being rich in microsatellite locus through a few step simple operations from the document enrichment with magnetic bead method of report, lowered experimental cost, shortened experimental period, and success ratio is high, is comparatively simple and effective at present SSR primer development method.

Kidney bean (Phaseolus vulgaris L.) is one of edible beans of cultivated area maximum in the world.Kidney bean originates in Central and South America, and the Kidney bean of Chinese cultivated is that the 15th century is directly introduced from America, and " Chinese " center " is considered to the secondary center of origin of Kidney bean, has abundant genetic diversity.Kidney bean is the important vegetable species of China, and cultivation history is long in China.Common kidney bean distributes the area extensively in China, and variety source is very abundant.Most of economize (city, the district) of China has Kidney bean to distribute, but mainly is distributed in provinces and regions such as Heilungkiang, the Inner Mongol, Shanxi, Shaanxi, Hubei, Sichuan, Guizhou, Yunnan.Nearly 120.51 ten thousand hectares of Chinese according to incompletely statistics common kidney bean sown area.

Carried out at present economical character and identified and enroll the Chinese common kidney bean resource that eats beans variety source catalogue have 4480 parts, these resources have been gone into national germplasm storehouse and have been preserved.M.R.Escribano etc. have carried out the genetic diversity result of study to 18 economical characters that are planted in 56 Kidney bean kinds in the 4 kinds of varying environments in the Spain northwestward and have shown between kind all proterties all there were significant differences and most proterties has significant gene environment to make effect mutually.The morphological markers of 4 short living kinds of 13 semi-wild kinds of 43 the cultivar CIATs in 60 Kidney bean variety source Heilongjiang Province and Poland having been carried out cluster analysis result during Luan Fei etc. is divided into two big types of short non-hibernating eggs with it and is one type and overgrows kind and the semi-wild kind is combined into one type.

The Zhang Chihong of vegetable or flower institute of the Chinese Academy of Agricultural Sciences etc. analyzes 10 economical characters of 324 parts of common kidney bean germ plasm resources; The result shows; China's common kidney bean germ plasm resource has abundant form variety; Average diversity index is 1.632, is higher than materials overseas, and utilizes 36 pairs of SSR primers that the genetic diversity of 332 parts of domestic common kidney beans, 16 parts of external common kidney beans and 29 parts of wild Kidney beans is analyzed.Xu Zhaosheng etc. have carried out the comprehensive characterization and evaluation in field to 38 parts of Kidney bean germplasms, to select the good excellent germplasm of comprehensive proterties, provide and produce and the scientific research utilization.Units such as the Chinese Academy of Agricultural Sciences Ins of Crop Varity Res, vegetable or flower institute, coastland, the Jiangsu Province institute of agricultural sciences, northeast agricultural university have carried out the collection of Kidney bean variety source; And resource carried out comprehensive evaluation; Think that China's common kidney bean germ plasm resource has abundant form variety, average diversity index is higher than materials overseas, Germplasm Resources on Phaseolus Vulgaris hereditary basis broad; Sibship is far away; Heritable variation mainly is present in the population inside of overgrowing, and Kidney bean becomes genetic diversity in population than horn of plenty, can be used as breeding material deposit germ plasm resource.And therefrom filter out the high elite germplasm material of strong, the tender pod protein contnt of a collection of disease resistance.

Molecular breeding comprises molecular marker assisted selection (MAS) and utilizes the genetic engineering means breeding.Molecular marker assisted selection is the key areas that molecular marking technique is used for crop improvement, is the product that traditional breeding technology and modern biotechnology combine.Have a lot of important characters (as output and the later stage leaf portion or fringe portion disease resistance etc.) only on ripe plant, just can show, therefore adopt traditional method in that after planting several months or several years all can not be selected it.And utilize molecule marker just can seedling (even to seed) to be detected, cultivate human and material resources and the financial resources that plant wasted thereby save greatly.

The SSR molecule marker has codominance, rich polymorphism in numerous molecule markers, numerous advantages such as wide distribute in the beans genome.At present just little satellite of known encoding sequence is screened and identify that little satellite of a large amount of non-coding regions is still waiting development and utilization.Therefore the present invention is that research object is opened the SSR primer of in the whole genome scope, developing more Kidney bean kinds with Chinese Kidney bean.

In sum, Kidney bean is very big in China's demand, in vegetables, occupies an important position; The Germplasm Resources on Phaseolus Vulgaris of China is abundant simultaneously; Genetic diversity is many, therefore is badly in need of the more SSR primer of exploitation, and molecular mark and conventional breeding are combined the process that could quicken the Kidney bean breeding; Cultivate new variety, realize society and economic benefit rapidly.

Summary of the invention

The objective of the invention is to be to provide a kind of preparation method of enrichment with magnetic bead method exploitation Kidney bean SSR primer; Utilize paramagnetic particle method to carry out the SSR primer development; Can screen the microsatellite sequence in the Kidney bean whole genome simply, apace; Can develop a large amount of SSR primers at short notice, have only after the SSR primer of exploitation some amount, just possibly carry out the analysis of SSR mark a certain species.Therefore, the present invention can provide guarantee for the realization of a large amount of SSR molecule marker of needs of work such as the Kidney bean construction of genetic atlas and the assignment of genes gene mapping.

A kind of preparation method of enrichment with magnetic bead method exploitation Kidney bean SSR primer comprises the following steps:

1. the extraction of Kidney bean genomic dna:

Choose Kidney bean kind with Kidney bean general feature.According to the cultivation management and the rich water measure plantation of routine, treat that seedling grows 5-6 sheet leaf, get tender leaf 0.4 gram and utilize the CTAB method to extract genomic dna.

Described Kidney bean, claim again kidney bean (be commonly called as two season beans or string bean), pulse family section bean.Kidney bean suits in temperate zone and the plantation of tropical high altitude localities, and more cold-resistant happiness light belongs to cross-pollination Kidney bean, short day crop, the kidney bean well developed root system, and leaf green, alternate, heart-shaped, flower is the leaf flower of worm, raceme, the long 15-18 of bennet centimetre.Blooming, to bear pods few more.It is nutritious, and protein contnt is high, promptly is that vegetables are again grain, also can make cake and beans filling, is the important agricultural byproducts of foreign exchange earning.Kidney bean formal name used at school Kidney bean, the Papilionaceae bean.

2. extract the detection of genomic dna concentration:

The concentration of extracting genomic dna detects with spectrophotometer.The genomic dna concentration that can carry out the SSR primer development will reach 100ng-1ug/ul.

3. enzyme is cut genomic dna:

With Mse I the Kidney bean genomic dna is carried out enzyme and cut, utilize AFLP amplification principle to cut pulsating two ends and add the single stranded oligonucleotide joint, and use with the supporting joint primer of joint and carry out the pcr amplification amplification, create genome PCR library, purifying is carried out in the library at enzyme.

4.Mse the I endonuclease bamhi adds joint:

Enzyme is cut the DNA product with Mse I joint (joint sequence: Oligo-MseI A5-TACTCAGGACTCAT-3 '; Oligo-Mse I B 5-GACGATGAGTCCTGAG-3 ') adding T4 DNAligase connects; Behind the mixing; Put 16 ℃ of thermostat water bath temperature and bathe and spend the night, be placed on then preserve in 4 ℃ of refrigerators subsequent use.

5. the enzyme PCR that cuts connection increases in advance:

Cutting the product that connects with step 4 enzyme is template, is that primer carries out pcr amplification with MseI-N:5 '-GATGAGTCCTGAGTAAN-3 '.Reaction conditions: 1 circulation of the first step, 94 ℃ of 5min, second step 18 circulations, 94 ℃ of 1min, 53 ℃ of 1min, 72 ℃ of 1min, 72 ℃ of 10min of the 3rd step 1 circulation, 4 ℃ of preservations.Reaction product uses the 1%W/V agarose electrophoresis to detect, and the dispersive region just occurs and gets final product, like Fig. 1.

6.PCR amplified production and biotinylated probes hybridization in advance:

Under suitable temperature (66-70 ℃), make PCR library and the biotin labeled repetition of being selected for use (2 bases repeat 10-15 time, and 3 or 4 bases repeat 5-8 time) sequence probe hybridize; Because the pan coating of magnetic bead has one deck Streptavidin; And this Streptavidin have very strong avidity with biology; Therefore biotin labeled probe with contain the unitary dna segment hybridization of Tumor-necrosis factor glycoproteins and combine after, just can be adsorbed by magnetic bead at an easy rate, fix through non-strict wash-out and strict wash-out (seeing below) and with magnetic field; Can remove other compositions of dna segment of not hybridized at an easy rate, thereby reach the purpose of highly enriched microsatellite sequence; The microsatellite DNA that under the sex change condition, contains Tumor-necrosis factor glycoproteins is by the TE buffer solution elution; The elutriant purifying is later as dna profiling.

Hybridization system TV is 100 μ L, and parallel altogether preparation 2 pipes (are respectively (AC) ₁₃(AG) ₁₃) packing then, hybridize.On the PCR appearance, carry out, response procedures is 95 ℃ of sex change 5min; The righttest hybridization temperature (66-70 ℃) keeps 1-2h down.In reaction system, add 300 μ L TEN100 solution after hybridization finishes, it is subsequent use that mixing is placed on 4 ℃ of preservations.The hybridization mixed solution that is added with 300 μ L TEN100 is joined through in the pretreated magnetic bead; (20-25 ℃ of room temperature; Below identical) go up to place 30min, during stir lightly frequently or piping and druming solution, thereby avoid the more effectively DNA after the absorption hybridization of magnetic bead deposition.(under room temperature, wash magnetic bead 3 times with 400 μ L TEN1000, each 5min stirs or the piping and druming mixture frequently then the magnetic bead of hybridizing to be carried out non-strict wash-out.Be preheated to 55 ℃ TEN1000 solution washing 1 time after 3 rinsings again with 400 μ L) and strict washing (with 400 μ L, 0.2 * SSC (3M NaCl, 0.3M Na ₃C ₆H ₅O ₇.2H ₂O; PH7.0, below identical)+0.1% (0.1g SDS, 100ml 2d H ₂O, below identical) SDS solution washs magnetic bead 3 times under room temperature, each 5min stirs or the piping and druming mixture frequently.Be preheated to 0.2 * SSC+0.1%SDS solution washing 1 time of 55 ℃ after three rinsings again with 400 μ L.With the fixing magnetic bead of magnet stand, remove washings).Add 50 μ L TE8.0, inhale with pipettor and beat evenly, then in 95 ℃ of following water-bath 5min.Stir frequently or the piping and druming mixture.With the fixing magnetic bead of magnet stand, the sucking-off supernatant is frozen in-20 ℃ rapidly, obtains target DNA fragment.With the eluted product is substrate, is primer with MseI-N, is used for pcr amplification with 5 μ L target DNA wash-out fragments behind the purifying, and amplification condition is consistent with the preparatory amplification condition of top PCR, 30 circulations of increasing.

7. connection transformed clone:

Pcr amplification product behind the purifying is connected on the T carrier clones.Concrete operations connect test kit (available from Promega) working instructions according to the pMD 18-T carrier that TaKaRa company provides.The ligation system is 10 μ L, and its formation is seen table 1.Condition of contact is 16 ℃ of 1hr.

Table 1DNA fragment and T carrier ligation system

In-70 ℃ of refrigerators, take out a pipe competent cell (M15,100 μ L), place ice bath to make it immediately and just melt (if the competent cell of made fresh can directly add and connect mixture).Add 5 μ L then and connect product, ice bath 30min.Ice bath finishes back 42 ℃ of heat shock 90sec, and ice bath 5min again adds the liquid LB substratum (not containing the ammonia benzyl) of 1mL precooling then, goes up recovery in 37 ℃ shaking tables (150rpm) and cultivates 45min.Recovery is cultivated and is finished the back in 4 ℃ of centrifugal 1min of 10000rpm, with sedimentation cell; Remove supernatant, residue 200-300 μ L substratum re-suspended cell.On each LB flat board, add 4 μ L IPTG and 40 μ L X-gal, coating is even, and per 100 μ L bacterium liquid are coated with a flat board, treats that liquid-absorbent finishes back (about 15min), is inverted in 37 ℃ spend the night (12-16hr) with flat board.

8. the picking of positive colony, cultivation, screening and order-checking:

Select positive colony with the order-checking of carrier universal primer, the pulsating dna sequence dna of the insertion that obtains is picked out suitable microsatellite DNA sequence.Cultured flat board (colony growth good and be evenly distributed) is placed some hrs at 4 ℃, bacterium colony is fully developed the color.Be cloned in 500 μ L (the containing 50 μ LAmp) liquid nutrient medium with aseptic toothpick picking white.With 200rmp rotating speed overnight cultures, preserve bacterium liquid for 4 ℃ on 37 ℃ of shaking tables.

Get 1 μ L bacterium liquid and be used for pcr template.Amplification condition is: 95 ℃ of 5min; 94 ℃ of 40s; 54 ℃ of 30s; 72 ℃ of 1min; Circulate 30 times; 72 ℃ of 7min.Get 2 μ L products electrophoresis on the 1%W/V sepharose, with full automatic gel imaging analysis system log (SYSLOG) electrophoresis result; Electrophoresis on the sepharose, and with full automatic gel imaging analysis system log (SYSLOG) electrophoresis result.

To contain the positive colony that inserts fragment and size to fit (450-800bp) gets half bacterium liquid and send Beijing big-and-middle living development in science and technology ltd's order-checking of China (Fig. 2).Second half adds the 20%V/V sterile glycerol, behind the mixing in-70 ℃ of frozen backups.Check order with the M13+ universal primer; Order-checking is carried out on ABI 3730XL DNA Sequencer; Used reaction reagent is ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit, and the useful length of every reaction can reach about 700-800bp.For the sequence that contains better repeated fragment, check order from other one section with the M13 primer, and forward and reverse sequencing result is spliced, to guarantee the accuracy of sequence.

9. the classification of sequential analysis and microsatellite locus and design:

With software SSRHunter (version 1.3.0; Li Qiang) from the sequence that obtains, finds out the microsatellite sequence that contains repeating unit.Instead wherein multiplicity more (2 bases repeat 10-15 time, 3 or 4 bases repetition 5-8 time), sequencing result is very good and the repeat region both wings have enough conserved regions sequence as microsatellite locus.Figure below shows the dna sequence dna (Fig. 3) and the order-checking peak shape figure (Fig. 4) thereof of an ideal microsatellite locus

The standard that proposes according to Weber (1990) is divided into 3 types with little satellite: perfect type (perfect), refer to not interrupt or near do not have the Tumor-necrosis factor glycoproteins of other kind Tumor-necrosis factor glycoproteins; Non-perfect type (imperfect), refer to 2 or above Tumor-necrosis factor glycoproteins of the same race by 3 non repetitive sequences below the base the interval; Compound (compound), the Tumor-necrosis factor glycoproteins that refers to a kind of Tumor-necrosis factor glycoproteins and other kind by the non repetitive sequence below 3 the interval.

Earlier with the microsatellite sequence of picking out with online tool VecScreen ( Http:// www.ncbi.nlm.nih.gov/VecScreen/) removal carrier sequence; Go out the position of Mse I joint (A and B) then in the sequence marked; Use primer-design software Primer Premier 5.0 (Rozen and Skaletsky 1988) between two end connectors and core repeat region, to design forward and inverse PCR amplimer again, be used for the pcr amplification in corresponding site.

10. micro-satellite primers validity check:

The validity of each site primer is closed polymorphum screen, obtain effective microsatellite locus.Two quality of random choose dna profiling are preferably carried out pcr amplification to all synthetic primers, and annealing temperature preferentially uses the default temperature of design during primer to be 50-62 ℃, if can not get amplified production or expanding effect is bad, then annealing temperature are adjusted.

Little satellite pcr amplification reaction system is 15 μ L, and it is constructed as follows table.Amplification condition is: 94 ℃ of 5min; 94 ℃ of 40s, Tm 30s, 72 ℃ of 40s, 35cycles; 72 ℃ of 7min.Pcr amplification product adds sample-loading buffer according to 5:1 with volume ratio, and 95 ℃ of sex change placed rapidly on ice after 5 minutes, and application of sample detects on 8% denaturing polyacrylamide gel then.In last appearance, add 0.1gPBR322/Msp I Marker, as the relative molecular weight standard of amplified production toward one of them well.The method dyeing tape reading of dying with silver at last.Result on polyacrylamide gel the electrophoretic result tape reading of the primer that can normally increase under the righttest annealing temperature (50-62 ℃), carry out pcr amplification with the dna profiling of picking out; The selection banding pattern is clear, has at least two or more allelic primer screenings to come out.Obtain 20 pairs of SSR primers that polymorphum is good altogether through this experiment.

Characteristics of the present invention: the enrichment with magnetic bead method be to utilize biotin labeling Tumor-necrosis factor glycoproteins specific probe and hybridize with the genomic dna endonuclease bamhi; Utilize vitamin H and the strong characteristic of streptavidin affinity again, carry out the enrichment that the Tumor-necrosis factor glycoproteins target fragment is accomplished in magnetic force absorption with the magnetic bead that encapsulates streptavidin.Through the cloning and sequencing amount being declined to a great extent behind the enrichment with magnetic bead and the segmental efficient that obtains to have Tumor-necrosis factor glycoproteins being increased substantially, therefore, become extremely effective means and being used widely rapidly of SSR primer development.Advantage of the present invention: just can obtain being rich in the dna fragmentation of microsatellite locus through a few step simple operations, lower experimental cost, shorten experimental period, and success ratio is high, is comparatively simple and effective at present SSR primer development method.The present invention altogether picking 105 positive colonies check order; Wherein there are 48 sequencing sequences can design primer; 20 pairs of effective primers from 48 pairs of primers, have been obtained; Effectively the primer yield be 19% (20/105) and Matthew W Blair etc. through genomic 120 the effective SSR primers that obtain to 3123 EST screening primers, effectively the primer yield is 3.8% (120/3123), this shows that developing SSR primer efficient of the present invention is high; And simple, can provide safeguard for large batch of developing SSR primer.

Description of drawings

Fig. 1 is a kind of thing synoptic diagram of expanding production in advance (the different cycle indexes of PCR).

" 1 " is cut the product that with joint be connected for different Kidney bean kinds through enzyme with " 2 " is template, is that primer carries out pcr amplification with MseI-N:5 '-GATGAGTCCTGAGTAAN-3 '; 14* wherein, 16*, 18*, 22* refer to PCR circulation 14 times, 16 times, 18 times, 22 times respectively.

Fig. 2 is a kind of bacterium liquid PCR product synoptic diagram.

Wells different among the figure are the result that primer carries out pcr amplification with M13 for being template with the different positive colonies of choosing.The band of choosing more than 500 checks order.

Fig. 3 is that a kind of AG repeats one of sequencing sequence.

This sequence is perfect sequence, and TC two bases repeat 13 times, and microsatellite sequence is between 474-499.

Fig. 4 is a kind of AG peak shape synoptic diagram that repeats to check order.

TC two bases repeat 13 times microsatellite sequence between 474-499, can design primer in its both sides, are exactly one of the SSR primer sequence that will develop of present method.

Embodiment

Below in conjunction with embodiment the present invention is done further explanation.

Embodiment 1:

A kind of preparation method of enrichment with magnetic bead method exploitation Kidney bean SSR primer the steps include:

A, extraction Kidney bean genomic dna carry out enzyme to genome and cut, and set up the AFLP-PCR library:

Kidney bean kind Qiao Yu (by Wuhan City vegetables Science Institute good Kidney bean kind is provided, overgrows, the leaf look green, and pattern is pale yellow).This kind is a fine Kidney bean kind, has the general feature of Kidney bean kind.According to the cultivation management and the rich water measure plantation of routine, treat that seedling grows 5-6 sheet leaf, get tender leaf 0.4 gram and utilize the CTAB method to extract genomic dna.

(1) adds 4ml 3 * CTAB extracting solution in the Eppeddorf of the 10ml pipe and (contain 0.2% beta-mercaptoethanol (W/V), preheating in 65 ℃ of water-baths.

(2) tender leaf of getting 0.4 gram Kidney bean is put in the mortar, adds suitable liquid nitrogen and repeatedly grinds to form fine powder rapidly, change in the CTAB extracting solution of preheating, with centrifuge tube lid tightly to prevent the beta-mercaptoethanol volatilization.Put back to insulation (65 ℃) 40min in the water-bath, light rolling keeps shaking up several times in the insulating process.

(3) take out the Eppenddorf pipe; Place on ice cool to room temperature rapidly, add isopyknic chloroform/primary isoamyl alcohol (24: 1), slightly mix and fully; Make it be mixed into milk sap up hill and dale; 4 ℃ of following centrifugal 12000rpm 10min draw 1ml with supernatant to be put in the 1.5ml centrifuge tube gently then, are put in-20 ℃ of refrigerators subsequent use.

(4) add 1 times of 100% (V/V) ethanol that volume is ice-cold among the DNA that extracts above.

(5) 4 ℃, behind the centrifugal 10min of 10000rpm, carefully outwell supernatant, the air-dry 5min of room temperature.

(6) add 1mlTE and 1 μ l RNase (10mg/ml), in 37 ℃, be incubated 40min.

(7) chloroform/the primary isoamyl alcohol extracting once to add equal-volume.The centrifugal 10min of 12000rpm draws supernatant and changes in the another one Eppendorf pipe, adds sodium-acetate and 2 times of absolute ethyl alcohols that volume is ice-cold of 1/10 volume, places 1h for-20 ℃.

(8) the centrifugal 10min of 10000rpm carefully outwells supernatant.

(9) the ethanol 1ml that adds 70% (V/V) washs the resuspended DNA of 150 μ l TE 3 times.

(10) be put in-20 ℃ of refrigerators and preserve.

The concentration of extracting genomic dna detects with spectrophotometer.With MseI the Kidney bean genomic dna is carried out enzyme and cut, endonuclease reaction is handled 20min with deactivation MseI enzyme for 65 ℃ then at PCR appearance or 37 ℃ of reactions of water-bath 3h.The reaction system (NEB buffer 2:2.0 μ L, 100 * NEB BSA:0.2 μ L, MseI 10U/ul:0.5 μ L, DNA:10 μ L, the ddH that join 20ul ₂O: mend to 20 μ L), enzyme is cut agarose and is detected.

Mse I joint sequence: Oligo-MseI A 5-TACTCAGGACTCAT-3 ', Oligo-MseI B5-GACGATGAGTCCTGAG-3 '.Preparation joint: Oligo-Mse I A and Oligo-Mse I B are made into 50 μ M stock solutions respectively; Fully after the dissolving; Take out each 50 μ L of equal-volume Oligo-MseI A and Oligo-MseI B; Fully behind the mixing in 94 ℃ of sex change 5min, slowly cool to room temperature after the taking-up, be stored in-20 ℃ subsequent use to concentration be 25uM.Linked system: total system 20ul (the 7ul enzyme is cut DNA product+1.5ul 10 * ligase buffer+4ul T4DNAl igase (3U/ul)+2.5ul MseI hybrid).Behind the mixing, put 16 ℃ of thermostat water bath temperature and bathe and spend the night, be placed on then preserve in 4 ℃ of refrigerators subsequent use.

The PCR that B, enzyme are cut connection increases in advance:

(1) Mse I-N primer sequence 5 '-GAT GAG TCC TGA GTA AN-3 ' (N=A, T, G, C) totally four pairs form mix primer.

(2) preparatory amplification reaction system

The amplified reaction TV is 20 μ L in advance, parallel altogether preparation 3 pipes.After the packing, carry out pcr amplification then.Amplified reaction constitutes like table 2:

Table 2 is amplification reaction system in advance

(3) the PCR condition that increases in advance: 94 ℃ of 5min; 94 ℃ of 1min, 53 ℃ of 1min, 72 ℃ of 1min; 72 ℃ of 7min.Design 3 groups of different cycle numbers (16/18/20cyles), every group differs 2 circulations, to confirm the optimization amplification condition.

(4) electrophoresis of amplified production check in advance

Get 3 μ LPCR reaction product, add 1 μ L sample-loading buffer, behind the mixing in the 1%W/V agarose gel electrophoresis.Successful sign is to occur a smear zone (200-800bp) in the electrophoresis product.The cycle number (18) that makes the smear zone just occur is best amplification cycles number.

C, the preparatory amplified production of PCR and biotinylated probes hybridization:

(1) hybridization system and condition

Hybridization system TV is 100 μ L, and parallel altogether preparation 2 pipes (are respectively (AC) ₁₃(AG) ₁₃) packing then, hybridize.Hybridization diagram of system 3:

Table 3 hybridization constitution system

On the PCR appearance, carry out, response procedures is 95 ℃ of sex change 5min; The righttest hybridization temperature (68 ℃) keeps 1h down.In reaction system, add 300 μ L TEN100 solution after hybridization finishes, it is subsequent use that mixing is placed on 4 ℃ of preservations.

(2) magnetic bead hybridization and washing

1. the pre-treatment of magnetic bead

With the 2mg magnetic bead with the careful washing of 300 μ L TEN100 solution 3 times, each 5min, when each washing finishes with the fixing magnetic bead of magnet stand, sucking-off supernatant.After washing finishes,, place 4 ℃ of preservations subsequent use with 40 μ L TEN100 solution suspension magnetic beads.

2. magnetic bead hybridization

The hybridization mixed solution that is added with 300 μ L TEN100 is joined through in the pretreated magnetic bead, places 30min on the room temperature, during stir lightly frequently or piping and druming solution, thereby avoid the more effectively DNA after the absorption hybridization of magnetic bead deposition.

3. magnetic bead washing

A. after magnetic bead absorption finishes,, remove the hybridization mixed solution with the fixing magnetic bead of magnet stand.

B. non-strict wash-out (nonstringency wash): under room temperature, wash magnetic bead 3 times with 400 μ L TEN1000, each 5min stirs or the piping and druming mixture frequently.Be preheated to 55 ℃ TEN1000 solution washing 1 time after 3 rinsings again with 400 μ L.

C. strict wash-out (stringency wash): under room temperature, wash magnetic bead 3 times with 400 μ L, 0.2 * SSC+0.1%SDS solution, each 5min stirs or the piping and druming mixture frequently.Be preheated to 0.2 * SSC+0.1%SDS solution washing 1 time of 55 ℃ after 3 rinsings again with 400 μ L.With the fixing magnetic bead of magnet stand, remove washings.

D. the wash-out of target DNA fragment: add 50 μ L TE8.0, with pipettor inhale beat even, then in 95 ℃ of following water-bath 5min.Stir frequently or the piping and druming mixture.With the fixing magnetic bead of magnet stand, the sucking-off supernatant is frozen in-20 ℃ rapidly.

D. the purifying of target DNA fragment and amplification:

With twice eluted product is substrate, is primer with MseI-N, is used for pcr amplification with 5 μ L target DNA wash-out fragments behind the purifying, and amplification condition is consistent with the preparatory amplification condition of top PCR, 30 circulations of increasing.

(1) carrier connects

Concrete operations connect the test kit working instructions according to the pMD 18-T carrier that TaKaRa company provides.The ligation system is 10 μ L, and it constitutes like table 4.Condition of contact is 16 ℃ of 1hr.

Table 4DNA fragment and T carrier ligation system

(2) conversion of connection product

The picking of E, positive colony, cultivation and screening:

(1) picking of positive colony, cultivation

A. general clone's bacterium colony that each is dull and stereotyped is controlled at about 200, conveniently to select positive colony.Cultured flat board (colony growth good and be evenly distributed) is placed some hrs at 4 ℃, make bacterium colony fully develop the color (positive colony is white, and empty plasmid be a blueness or light blue).

B. be cloned in 500 μ L (the containing 50 μ LAmp) liquid nutrient medium with aseptic toothpick picking white.

C.37 on ℃ shaking table with 200rmp rotating speed overnight cultures, preserve the bacterium liquid for 4 ℃

(2) PCR check positive colony

Get 1 μ L bacterium liquid and be used for pcr template.Amplification reaction system is 15 μ L, does negative control simultaneously, and reaction system constitutes sees table 5.Amplification condition is: 95 ℃ of 5min; 94 ℃ of 40s; 54 ℃ of 30s; 72 ℃ of 1min; Circulate 30 times; 72 ℃ of 7min.

Table 5 is used to detect the PCR reaction system of positive colony

Get 2 μ L products electrophoresis on 1% sepharose, and with full automatic gel imaging analysis system log (SYSLOG) electrophoresis result.To contain the positive colony that inserts fragment and size to fit (450-800bp) gets half bacterium liquid and send the order-checking of Beijing China big-and-middle living development in science and technology ltd.Second half adds 20% sterile glycerol, behind the mixing in-70 ℃ of frozen backups.Check order with the M13+ universal primer; Order-checking is carried out on ABI 3730XL DNA Sequencer; Used reaction reagent is ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit, and the useful length of every reaction can reach about 700-800bp.For the sequence that contains better repeated fragment, check order from other one section with the M13 primer, and forward and reverse sequencing result is spliced, to guarantee the accuracy of sequence.

The classification of F, sequential analysis and microsatellite locus and design:

With software SSRHunter (version 1.3.0; Li Qiang) from the sequence that obtains, finds out the microsatellite sequence that contains repeating unit.Earlier the microsatellite sequence of picking out is removed the carrier sequence with online tool VecScreen; Go out the position of Mse I joint (A and B) then in the sequence marked; Use primer-design software Primer Premier 5.0 (Rozen and Skaletsky again; 1988) between two end connectors and core repeat region, design forward and inverse PCR amplimer, be used for the pcr amplification in corresponding site.When the design primer, (primer length is between 18-27bp, and optimal length is 20bp for relevant parameter inaction default value; The amplification annealing temperature is between 50-63 ℃, and optimum temperuture is 60 ℃; GC content (GC%) is between 20-80%, and optimum content is 50%; Amplified production length is between 100-500bp, and optimal length is 200bp).

G, micro-satellite primers validity check:

(1) little satellite pcr amplification

Two quality of random choose dna profiling are preferably carried out pcr amplification to all synthetic primers, and annealing temperature preferentially uses the default temperature of design during primer to be 50-62 ℃, if can not get amplified production or expanding effect is bad, then annealing temperature are adjusted.

Little satellite pcr amplification reaction system is 15 μ L, and it constitutes like table 6.Amplification condition is: 94 ℃ of 5min; 94 ℃ of 40s, Tm 30s, 72 ℃ of 40s, 35cycles; 72 ℃ of 7min.

The little satellite amplification reaction system of table 6

(2) pcr amplification product detects

Pcr amplification product added sample-loading buffer according to 5: 1 with volume ratio, and 95 ℃ of sex change placed rapidly on ice after 5 minutes, and application of sample detects on 8% denaturing polyacrylamide gel then.In last appearance, add 0.1g PBR322/Msp I Marker, as the relative molecular weight standard of amplified production toward one of them well.The method dyeing tape reading of dying with silver at last.The concrete operations step is following:

1. wash sheet glass.With brush sheet glass is cleaned oven dry, clean 3 times with alcohol again before the encapsulating.

2. silication and anti-silication sheet glass.The silication agent for preparing (200 μ L peel off silane+2mL 95% ethanol) is poured on the short slab, even with the rapid wiping of filter paper; The anti-silication agent that configures (the affine silane of 9 μ L+18 μ L glacial acetic acid+2mL, 95% ethanol) is poured on the long slab, even with the rapid wiping of filter paper.In ventilating kitchen, dried then 15 minutes.

3. encapsulating.Sheet glass is installed, tilt to be put in slightly on the flat desktop.In a small beaker, add the urea gel monomer that 60mL configures, 400 μ L 10%AP shake up slowly encapsulating of back behind the 40 μ L TEMED, avoid occurring bubble.

4. gel.The offset plate horizontal positioned, the anti-comb that inserts was placed 3 hours.

5. prerunning.Comb is extracted, cleaned offset plate and comb, on electrophoresis chamber, install offset plate, last groove is respectively irritated 1 * TBE 400mL with following groove.The permanent power electrophoresis of 45w 1 hour to 45 ℃.

6. go up the appearance electrophoresis.Stop prerunning earlier before the last appearance, will go up a kind top blast wash clean, insert good comb then with rifle.Every hole point sample 3 μ L, 45w electrophoresis 1.5 hours.

7. silver dyes.The first step is pulled down the acetum of putting into 1.5L10% with long slab and is fixed 30 minutes; Second step, remove stationary liquid, with rinsed with deionized water 3 times, each two minutes; In the 3rd step, dyeing is 30 minutes in containing 1 ‰ Silver Nitrate dye liquors of 1.5 ‰ formaldehyde solutions; The 4th step, rinsed with deionized water 10 seconds; In the 5th step, developing solution (3% sodium carbonate solution) colour developing is clear to being with; The 6th goes on foot, and stationary liquid is poured in the colour developing liquid stopped showing.

8. system for photographing.With digital camera the offset plate that developed the color is taken a picture.

(3) screening in polymorphic micro-satellite site

Result on polyacrylamide gel the electrophoretic result tape reading of the primer that can normally increase under the righttest annealing temperature, carry out pcr amplification with the dna profiling of picking out; The selection banding pattern is clear, has at least two or more allelic primer screenings to come out.

Claims

1. the preparation method of an enrichment with magnetic bead method exploitation Kidney bean SSR primer comprises the following steps:

The extraction of A, Kidney bean genomic dna:

Choose the Kidney bean kind, according to the cultivation management and the rich water measure plantation of routine, seedling grows 5-6 sheet leaf, gets tender leaf 0.4 gram and utilizes the CTAB method to extract genomic dna;

B, the detection of extracting genomic dna concentration:

The concentration of extracting genomic dna detects with spectrophotometer, and the genomic dna concentration of carrying out the SSR primer development will reach 100ng-1ug/ul;

C, enzyme are cut genomic dna:

With Mse I the Kidney bean genomic dna is carried out enzyme and cut, utilize AFLP amplification principle to cut pulsating two ends and add the single stranded oligonucleotide joint, and use with the supporting joint primer of joint and carry out the pcr amplification amplification, create genome PCR library, purifying is carried out in the library at enzyme;

D, Mse I endonuclease bamhi add joint:

Enzyme is cut the DNA product with Mse I joint: joint sequence Oligo-MseI A5-TACTCAGGACTCAT-3 '; Oligo-Mse I B 5-GACGATGAGTCCTGAG-3 ' adds T4 DNAligase and connects; Behind the mixing; Put 16 ℃ of thermostat water bath temperature and bathe and spend the night, be placed on then preserve in 4 ℃ of refrigerators subsequent use;

The PCR that E, enzyme are cut connection increases in advance:

Cutting the product that connects with step D enzyme is template, is that primer carries out pcr amplification, reaction conditions with MseI-N:5 '-GATGAGTCCTGAGTAAN-3 ': 1 circulation of the first step; 94 ℃ of 5min, second step 18 circulations, 94 ℃ of 1min; 53 ℃ of 1min, 72 ℃ of 1min, 72 ℃ of 10min of the 3rd step 1 circulation; 4 ℃ of preservations, reaction product use the 1%W/V agarose electrophoresis to detect;

F, the preparatory amplified production of PCR and biotinylated probes hybridization:

Under 66-70 ℃, make PCR library and the biotin labeled repetitive probe of being selected for use hybridize; The pan coating of magnetic bead has one deck Streptavidin, biotin labeled probe with contain the hybridization of the unitary dna segment of Tumor-necrosis factor glycoproteins and combine after, fix through washing process and with magnetic field, the microsatellite DNA that under the sex change condition, contains Tumor-necrosis factor glycoproteins is by the TE buffer solution elution; The elutriant purifying is later as dna profiling;

Hybridization system TV is 100 μ L, and parallel altogether preparation 2 pipe packing are hybridized, and on the PCR appearance, carry out, and response procedures is 95 ℃ of sex change 5min; Hybridize 66-70 ℃ and keep 1-2h down, in reaction system, add 300 μ L TEN100 solution after hybridization finishes, it is subsequent use that mixing is placed on 4 ℃ of preservations, and the hybridization mixed solution that is added with 300 μ L TEN100 is joined through in the pretreated magnetic bead; Place 30min on the room temperature, the magnetic bead to hybridization carries out wash-out then, and SDS solution washs magnetic bead 3 times under room temperature; Each 5min is preheated to 0.2 * SSC+0.1%SDS solution washing 1 time of 55 ℃ again with 400 μ L after three rinsings, add 50 μ L TE8.0; Beat evenly with the pipettor suction, in 95 ℃ of following water-bath 5min, frozen then in-20 ℃; Obtaining target DNA fragment, is substrate with the eluted product, is primer with MseI-N; 5 μ L target DNA wash-out fragments with behind the purifying are used for pcr amplification, and amplification condition is consistent with the preparatory amplification condition of top PCR, 30 circulations of increasing;

G, connection transformed clone:

Pcr amplification product behind the purifying is connected on the T carrier clones, operation connects according to pMD 18-T carrier, and the ligation system is 10 μ L; Condition of contact is 16 ℃ of 1hr, in-70 ℃ of refrigerators, takes out a pipe competent cell, places ice bath to melt; Add 5 μ L and connect product, ice bath 30min, ice bath finish back 42 ℃ of heat shock 90sec; Ice bath 5min again adds the liquid LB substratum of 1mL precooling then, and 45min is cultivated in recovery on 37 ℃ shaking table; Recovery is cultivated and is finished the back in 4 ℃ of centrifugal 1min of 10000rpm, with sedimentation cell; Remove supernatant, residue 200-300 μ L substratum re-suspended cell adds 4 μ LIPTG and 40 μ L X-gal on each LB flat board, and coating is even, and per 100 μ L bacterium liquid are coated with a flat board, treat that liquid-absorbent finishes after, flat board is inverted in 37 ℃ spends the night;

The picking of H, positive colony, cultivation, screening and order-checking:

Selecting positive colony checks order with the carrier universal primer; The pulsating dna sequence dna of the insertion that obtains is picked out the microsatellite DNA sequence, with cultured flat board 4 ℃ of placements; Make the bacterium colony colour developing; Be cloned in the 500 μ L liquid nutrient mediums with aseptic toothpick picking white, with 200rmp rotating speed overnight cultures, preserve bacterium liquid for 4 ℃ on 37 ℃ of shaking tables;

Get 1 μ L bacterium liquid and be used for pcr template, amplification condition is: 95 ℃ of 5min; 94 ℃ of 40s; 54 ℃ of 30s; 72 ℃ of 1min; Circulate 30 times; 72 ℃ of 7min.Get 2 μ L products electrophoresis on the 1%W/V sepharose, with full automatic gel imaging analysis system log (SYSLOG) electrophoresis result;

To contain the positive colony that inserts fragment and size and get half bacterium liquid order-checking; Second half adds 20%V/V bacterium glycerine; In-70 ℃ of frozen backups, with the order-checking of M13+ universal primer, order-checking is carried out on ABI 3730XLDNA Sequencer behind the mixing; Used reaction reagent is ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit; The useful length of every reaction reaches 700-800bp, checks order from other one section with the M13 primer, and forward and reverse sequencing result is spliced;

The classification of J, sequential analysis and microsatellite locus and design:

From the sequence that obtains, find out the microsatellite sequence that contains repeating unit with software SSRHunter, order-checking, the standard that proposes according to Weber is divided into 3 types with little satellite: perfect type, refer to not interrupt or near do not have the Tumor-necrosis factor glycoproteins of other kind Tumor-necrosis factor glycoproteins; Non-perfect type, refer to 2 or above Tumor-necrosis factor glycoproteins of the same race by 3 non repetitive sequences below the base the interval; Compound; The Tumor-necrosis factor glycoproteins that refers to a kind of Tumor-necrosis factor glycoproteins and other kind by the non repetitive sequence below 3 the interval; The microsatellite sequence of picking out is removed the carrier sequence; Go out the position of Mse I joint in the sequence marked, between two end connectors and core repeat region, design forward and inverse PCR amplimer with primer-design software Primer Premier 5.0 again, be used for the pcr amplification in corresponding site;

K, micro-satellite primers check:

The validity of each site primer is closed polymorphum screens; Obtain effective microsatellite locus; Select two dna profilings all synthetic primers are carried out pcr amplification; Default temperature when annealing temperature is used the design primer is 50-62 ℃, and little satellite pcr amplification reaction system is 15 μ L, and amplification condition is: 94 ℃ of 5min; 94 ℃ of 40s, Tm 30s, 72 ℃ of 40s, 35cycles; 72 ℃ of 7min; Pcr amplification product adds sample-loading buffer according to 5:1 with volume ratio; 95 ℃ of sex change were placed on ice in 5 minutes; Application of sample detects on 8% denaturing polyacrylamide gel, in last appearance, adds 0.1gPBR322/Msp I Marker toward one of them well, and the primer of normal amplification reads tape to the result electrophoretic result on polyacrylamide gel who under 50-62 ℃ of annealing, carries out pcr amplification with the dna profiling of picking out; The selection banding pattern is clear, has at least two or more allelic primer screenings to come out.