CN101985655A - Buchloe dactyloides ISSR-PCR molecular marker system - Google Patents

Abstract

The invention belongs to the technical field of buchloe dactyloides molecular breeding, and particularly relates to an optimal buchloe dactyloides ISSR-PCR (inter-simple sequence repeat-polymerase chain reaction) reaction system. The invention provides the following ISSR-PCR reaction system that: each 25muL of reaction system contains 1*PCR buffer solution, 0.3mM of dNTPs, 1.0U of Taq enzyme, 0.6muM of any one of primers shown as SEQ ID 1-7, 2.0mM of Mg2+ and 50ng of DNA template; and the reaction mixed solution is amplified by the following procedures of: pre-denaturing for 3 minutes at 94 DEG C, denaturing for 30 seconds at 94 DEG C, annealing for 30 seconds at 50 to 60 DEG C, stretching for 2 minutes at 72 DEG C, circulating for 35 times, and stretching for 4 minutes at 72 DEG C. The buchloe dactyloides ISSR-PCR provided by the invention has good stability and high definition, and increases the richness of strips.

Description

A kind of buffalograss ISSR-PCR molecule marker system

Technical field

The invention belongs to buffalograss molecular breeding technical field, be specifically related to a cover buffalograss ISSR-PCR and react best system.

Background technology

Buffalograss (Buchloe dactyloides) is the perennial lawn of Gramineae Herba Eragrostidis pilosae subfamily buffalo grass grass, it mainly is taken as pasture growing in nearly 100 years, but since its have a lot of good characteristics and preferably the level ground be worth (as drought-enduring, plant is short, the very thin softness of blade etc.) and be used to planting lawn gradually, and compare not tangible disease with other turf grass species.Along with the worsening shortages of water resources and the public pay attention to day by day to environmental requirement, the buffalograss lawn is paid attention to by people gradually owing to have very strong drought tolerance.Because its superior drought resistance and adaptability become one of main grass seeds of regional afforestation such as China North China, northeast, northwest, environment protection, soil conservation.At present the buffalograss breeding technique adopts remains methods such as traditional selection breeding, selection by mutation, make that the legacy basis of buffalograss kind is narrow day by day, proterties that some are important such as drought tolerance, quality, thermotolerance etc. all have losing in various degree, thereby have limited the development of China's buffalograss breeding industry.

Molecular marker assisted selection (molecular maker-assisted selection) is the molecule marker according to objective trait, a kind of breeding method that the breeding offspring is selected, its ultimate principle is to utilize the molecule marker that is divided into from relation with target gene close linkage or performance to carry out target area and full genome screening to selecting individuality, thereby reduce chain burden, obtain the individuality of expectation, reach the purpose that improves breeding efficiency.Because molecule marker is not subjected to the influence of envrionment conditions, the selection that can fast and effeciently select the individual plant that has objective trait, the polymerization of accelerating excellent genes and improve the crop quantitative character, thereby this method can improve the accuracy that breeding is selected, shortening breeding cycle, quickening breeding process.

Sequence mark (ISSR) between simple iteron is to be the PCR molecule marker of primer with little satellite, and it is directly analyzed genomic dna, is not subjected to any environmental influence.It is a primer with the microsatellite DNA of grappling, promptly 3 ' end or the 5 ' end in the SSR sequence adds 2～4 random nucleotides, having only on genome could be fixed by target with the site of the Nucleotide of grappling coupling, avoid the slip of SSR in genome, has improved the specificity of pcr amplification widely.ISSR primer development expense is low, and is simple to operate, can disclose higher polymorphism than RAPD and RFLP.It is identified in the structure of herbage genetic linkage maps, the assignment of genes gene mapping, germ plasm resource, herbage is evolved and aspects such as the analysis of genetic diversity, molecular marker assisted selection obtain more and more widely application.The key of ISSR molecule marker test is the optimization to reaction system and test conditions.The ISSR-PCR system of different materials exists very big-difference, the consumption of the concentration of dna profiling and purity, primer and dNTP, Taq enzyme and marque, Mg ²⁺Concentration, all multifactor results that all can influence test such as the concentration of primer, reaction times and pH value size.

Livestock industry has in recent years had very great development, and the ISSR labeling technique is used in herbage research in succession, but at present both at home and abroad about the report of buffalograss ISSR labeling technique also seldom, has only utilization ISSR such as Liu Li to study the buffalograss multifarious phenotype of colony of growing directly from seeds.Its selected ISSR-PCR reaction system is (20 μ L): 1 * PCR buffer damping fluid, 0.2mM dNTPs, 1.0U Taq enzyme, 0.5 μ M primer, 2.0mM Mg ²⁺, dna profiling 30ng, sharpness and a bit deficient in of band richness.

In general, the also very big gap of existence is compared in the research of buffalograss resource with other herbage.Therefore set up the scientific and reasonable ISSR-PCR reaction system of buffalograss, seek best theoretical combination, both can accelerate the test process, the foundation and the directive function of science is provided for follow-up work, can improve the efficient of molecular mark again, shortening the breeding cycle.

Summary of the invention

The purpose of this invention is to provide a kind of buffalograss ISSR-PCR reaction system and application thereof.

Wherein, contain 1 * PCR damping fluid in the reaction system of per 25 μ L, dNTPs 0.3mM, Taq enzyme 1.0U, the arbitrary primer 0.6 μ M in SEQ IDNo.1～7 primers, Mg ²⁺2.0mM, dna profiling 50ng; Above-mentioned reaction mixture is increased by following program:

94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30s, 50～60 ℃ of annealing 30s, 72 ℃ are extended 2min, circulate 35 times, and 72 ℃ are extended 4min again.

Preferred annealing temperature is determined according to selected primer, and is specific as follows:

SEQ?ID?No.1?52.8℃

SEQ?ID?No.2?49.9℃

SEQ?ID?No.3?57.7℃

SEQ?ID?No.4?49.9℃

SEQ?ID?No.5?57.7℃

SEQ?ID?No.6?48.8℃

SEQ?ID?No.7?57.7℃。

Buffalograss ISSR-PCR reaction system rich polymorphism provided by the invention, background is clear and signal is strong.

The present invention has carried out the foundation and the optimization work of buffalograss ISSR-PCR reaction system comparatively meticulously, has determined the ISSR-PCR optimum response system of suitable buffalograss, has improved the accuracy and the stability of test greatly, has shortened the breeding time limit.The present invention is labeled as starting point with ISSR, remedied the deficiency of the buffalograss genetic diversity being studied both at home and abroad, the invention achievement can be used for aspects such as buffalograss cross-breeding, genetic affinity, molecular mark, breeding of new variety, has very big scientific value and using value.

Description of drawings

Fig. 1 is ISSR-PCR exploration orthogonal test figure.Wherein M represents DL2000 Marker; 1-9 represents treatment combination 1-9.

Fig. 2 is ISSR-PCR fine tuning orthogonal test figure.Wherein M represents DL2000 Marker; 1-9 represents treatment combination 1-9.

Two kinds of reaction system results' of Fig. 3 comparison.Wherein M represents DL2000 Marker; 1-9 represents treatment combination 1-9.

Fig. 4 is determining of primer TP10 optimum annealing temperature.

Embodiment

Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.

If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.

Embodiment 1 buffalograss extracting genome DNA

(1) get about fresh buffalograss blade 1g, clear water is cleaned, is dried, and shreds to put into mortar, pours liquid nitrogen cooling, grinding subsequently into;

(2) powder after will grinding adds 65 ℃ 2 * CTAB extracting solution, 900 μ L, 65 ℃ of water-bath 30min;

(3) the cooling back adds 500 μ L chloroform/primary isoamyl alcohol (volume ratio 24: 1);

(4) centrifugal 10min under the 8000rpm;

(5) get supernatant liquor;

(6) repeat (4), (5) once;

(7) 3M sodium-acetate and isopyknic Virahol of adding 1/10 volume shake up, and place on ice more than the 5min to flocks occurring;

(8) centrifugal 15min under the 12000rpm outwells supernatant liquor;

(9) use 75% alcohol wash, it is standby to be dissolved in 4 ℃ of preservations of TE damping fluid behind the drying at room temperature 1h.

The foundation and the optimization of embodiment 2 ISSR-PCR quadrature systems (25 μ L)

According to the orthogonal design principle, adopt L ₉(3 ⁴) orthogonal array, at first to dNTPs, Taq enzyme, primer and Mg ²⁺Concentration is carried out the exploratory orthogonal test of 4 factors, 3 levels, and exploratory orthogonal array sees Table 1; According to the result of exploratory orthogonal test, dwindle the concentration gradient of each primer and carry out the fine tuning orthogonal test again, the fine tuning orthogonal design sees Table 3.Selected ISSR-PCR primer sequence is 5 '-AGAGAGAGAGAGAGAGYG-3 ' [Y=(C, T)], 2 repetitions are established in test.The ISSR-PCR amplification program: 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 2min, circulate 35 times, and 72 ℃ are extended 4min again, last 4 ℃ of preservations.

2.1 the foundation of exploratory ISSR-PCR reaction system

The orthogonal experimental design of exploration ISSR-PCR reaction system sees Table 1.

Exploratory orthogonal experimental design [the L of table 2 ₉(3 ⁴)] unit: μ L

The exploration orthogonal experiments is seen Fig. 1, and No. 1 and No. 2 processing does not amplify band.Handle for No. 3 very faint band is arranged.But it is not high to handle the more sharpness of band No. 4 and No. 8, illustrates that its amplification efficiency is not very high.The band difference of handling between two repetitions for No. 6 and No. 9 illustrates that its stability is good inadequately.No. 5 and No. 7 treatment combination bands are many and very clear, repeatability might as well, illustrate that apart from the optimum combination deviation be basic point with it not quite again, dwindle the concentration gradient of each factor and carry out the fine tuning orthogonal test.

2.2 the foundation of fine tuning ISSR-PCR reaction system

The orthogonal experimental design of fine tuning ISSR-PCR reaction system sees Table 2.

Table 2 fine tuning orthogonal experimental design [L ₉(3 ⁴)] unit: μ L

The fine tuning orthogonal experiments is seen Fig. 2, and except No. 9 were handled, other 8 treatment combinations had all been run out of tangible band.1,3, No. 7 treatment combinations have amplified than multi-ribbon but 2 reproducible results are not quite alike, and this 3 reaction system less stables are described.It is more weak not too clear that but No. 5 treatment combinations expand the more band of the band that.Handle for No. 4 to expand and 3 bands, good reproducibility, but the band color is shallow slightly.No. 6 treatment combinations only expand and 2 very light bands.The band of handling for No. 8 seldom, repeatability is also bad.What test effect was best is No. 2 treatment combinations, amplified 5 bands altogether and also band is clear, repeatability might as well, be the treatment combination of the best.

The fine tuning orthogonal experiments is measured the relative concentration value of each band with Quantity One software in the BIORAD Gel Doc-XR gel imaging system, carry out the quadrature intuitive analysis then, as shown in table 3.

The intuitive analysis of table 3 ISSR-PCR fine tuning orthogonal experiments

Embodiment 3 ISSR-PCR reaction system Detection of Stability

Select other template DNA for use, use the optimum handling combination on best combination in 9 treatment combinations of fine tuning orthogonal test (No. 2 handle) and the statistical theory to carry out ISSR-PCR respectively, detect the stability of 2 individual system and compare the amplification efficiency of 2 individual system.

The result as shown in Figure 3.These 2 amplification system bands are the same substantially, but some band that best theoretical combination amplifies is obviously brighter, and the amplified band number is more, so select for use best theory to be combined as the ISSR-PCR reaction system of official test.So select for use best theory to be combined as the ISSR-PCR reaction system of official test, promptly contain 1 * PCR damping fluid in the reaction system of 25 μ L, dNTPs 0.3mM, Taq enzyme 1.0U, primer 0.6 μ M, Mg ²⁺2.0mM, dna profiling 50ng.

Determining of embodiment 4 primer screenings and optimum annealing temperature

This test ISSR is provided by the sequence that primer adopts Canadian Columbia University (UBC) to provide, and sees Table 4 by Shanghai 33 primer information of living worker's synthetic.

Determine on the optimum response system basis in test, on U.S.'s grads PCR instrument (Thermo), carry out determining of primer screening and optimum annealing temperature.Primer that filters out and optimum annealing temperature information thereof see Table 5.Test is provided with 12 annealing temperatures: 48.0 ℃, and 48.1 ℃, 48.8 ℃, 49.9 ℃, 51.3 ℃, 52.8 ℃, 54.5 ℃, 56.1 ℃, 57.7 ℃, 59.0 ℃, 59.9 ℃, 60.5 ℃.

The relevant information of the selected primer of this test of table 4

Annotate: Y-pyrimidine, R-purine

Table 5 primer screening result

Good system increases with 33 primers respectively to adopt screening, and the result shows that the band that above 7 primer amplifications come out is bright, and signal is strong and polymorphism good.The band that most of materials adopt the agaroses amplification all 5 and more than, and polymorphism information content is abundant.

With the TP10 primer is example, and as seen from Figure 4, when annealing temperature was relatively lower, but the effect band of ISSR-PCR amplification was a lot of very fuzzy, illustrated that the specificity of ISSR-PCR amplification when annealing temperature is low is bad; When annealing temperature than the band of higher time amplification less and band also a little less than, illustrate that amplification efficiency is not high.When annealing temperature at 49.9 ℃ time amplification more and band is clear.Therefore the optimum annealing temperature of determining this primer is 49.9 ℃.In like manner, the optimum annealing temperature of other primers is determined by this principle.

Claims (4)

1. a buffalograss ISSR-PCR reaction system is characterized in that, contains 1 * PCR damping fluid in the reaction system of per 25 μ L, dNTP 0.3mM, Taq enzyme 1.0U, primer 0.6 μ M, Mg ²⁺2.0mM, dna profiling 50ng; Above-mentioned reaction mixture is increased by following program:

94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30s, 50～60 ℃ of annealing 30s, 72 ℃ are extended 2min, circulate 35 times, and 72 ℃ are extended 4min again.

2. ISSR-PCR system according to claim 1 is characterized in that, described primer is selected from arbitrary in SEQ ID No.1～7.

3. ISSR-PCR system according to claim 1 is characterized in that, described annealing temperature is determined according to selected primer, and is specific as follows:

SEQ?ID?No.1?52.8℃

SEQ?ID?No.2?49.9℃

SEQ?ID?No.3?57.7℃

SEQ?ID?No.4?49.9℃

SEQ?ID?No.5?57.7℃

SEQ?ID?No.6?48.8℃

SEQ?ID?No.7?57.7℃。

4. according to the application of each described ISSR-PCR system of claim 1～3 in evaluation of buffalograss genetic diversity and molecular mark.

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