CN1428440A - Molecular marker linked with wheat mildew-resistance gene - Google Patents
Molecular marker linked with wheat mildew-resistance gene Download PDFInfo
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Abstract
The present invention relates to a molecular marker linked with wheat anticalcino gene and its obtaining method. Said invention adopts two molecular marking methods of RAPD and SSR to make screening of resistance marker for wheat parent and nosotropic filial generation "Brock/015//Jing 411", in which RAPD marker uses 10bp oligonucleotide developed by American OPERON co. as random primer, and SSR marker uses the primer synthesized by SSR primer sequence reported by R & der in 1998, after screening, the molecular marker OPP15900 and Xgwm 114120 linked with wheat anticalcino gene can be obtained. These markers can be used for auxiliary selection breeding of anticalcino wheat, at the same time can lay the foundation of cloning new anticalcino gene.
Description
Technical field: the invention belongs to agricultural biotechnology engineering, particularly linked molecule marker and preparation method thereof with powdery mildew resistance gene.
Technical background:
The wheat powdery mildew that is caused by obligatory parasitism fungi wheat powdery mildew (Erysiphe graminis f.sp.tritici) is one of main disease of harm Wheat Production.In recent years, along with popularization of short stem, the semi-dwarf mutant wheat breed, the increase of field cultivation density and the improvement of water and fertilizer condition, this disease is on the rise in China's north and south wheat district hazard rating, becomes one of major obstacle factor of current improving yield of wheat, stable yields.Nineteen ninety, wheat powdery mildew was popular in 39% wheat planting district, the whole nation, caused production loss to reach 400,000,000 kilograms.At present, wheat powdery mildew has become one of common disease that threatens China's Wheat Production, so very urgent to the control of this disease.
Although can take chemical agent to prevent and treat wheat powdery mildew in the practice,, cultivate disease-resistant variety and be the safest, economy, effective measures.At the beginning of the seventies, China introduces the wheat derived varieties of tool 1B/1R as anti-source, is once once having the kind more than 90% to carry the Pm8 mildew-resistance gene, because the unicity of resistant gene, the result causes wheat powdery mildew repeatedly to be very popular in China.After this, China wheat breeding man is devoted to introduce in many ways the kind that has new disease-resistant gene with transformation, but because some disease-resistant gene is expressed inconsistent in different genetic backgrounds, cause and really be used to produce actual mildew-resistance gene seldom, that uses in breeding only has a Pm2, Pm4a, Pm4b, Pm6, Pm8 and combination Pm2+6 thereof, Pm2+4 etc., adding wheat powdery mildew, to have a colony big, wide accommodation, characteristics such as the many and virulence variation of physiological strain is fast, the firm application soon of disease-resistant variety (being) that makes many costs time cultivation for many years, that have even also use, just owing to germ colony changes of toxicity has been lost resistance.The enforcement period of the ninth five-year plan, Pm4a, Pm6 have all begun to lose resistance throughout the country.China first transformation success and Pm21 gene virose bacterial strain of naming have voluntarily been released in twentieth century end, Beijing area.At present, utilized this gene comparatively widely in China's wheat breeding project, should cause vigilantly, prevented that the single phenomenon in anti-source from taking place once more, controls the popular of Powdery Mildew effectively.Must excavate and utilize for this reason new resistance source energetically and constantly carry out adding up of disease-resistant gene, cultivate the persistent wheat breed of resistance.
For realizing above-mentioned task, excavate new resistance source, in the past, breeding men came assistant breeding by morphology mark and biochemical marker mostly, but this class mark has shortcomings such as workload is big, mark quantity is few, easy affected by environment.Along with development of molecular biology, a class is applied to wheat anti-powdery mildew research by domestic and international many scholars based on the molecular marking technique of DNA variation.Use molecule marker, can exempt heavy work program in the traditional breeding method, follow the tracks of, detect foreign gene, can also be aggregated to a plurality of mildew-resistance genes in the same improved seeds, obtain durable resistance.In addition, gene accurately being positioned on the highdensity molecular linkage map, is starting point with closely linked molecule marker, uses karyomit(e) walking methods such as (Chromosome Walking), approaches target gene gradually, finally clones this gene.Facts have proved that dna molecular marker is to carry out the important tool that disease resistance is identified and assistant breeding is studied.
Wheat is mainly controlled by major gene the resistance of Powdery Mildew, and the early gene symbol is M1, and the gene symbol of definite designation is Pm (powdery mildew).Since nineteen thirty Australia scholar reported first since Australian spring wheat variety Thew carries a dominance mildew-resistance gene, so far 30 Pm gene locuss have been identified, except that Pm29, all the other major genes have been positioned on karyomit(e) or the chromosome arm.Disease-resistant gene is still arranged in addition not by definite designation, represent with M1 temporarily.Since carrying out the powdery mildew resistance gene in wheat molecule marking research beginning of the nineties, existing disease-resistant gene near half has found corresponding molecule marker.The molecule marker that has obtained at present some owing to the variation of powdery mildew physiological strain was lost efficacy, resistant gene is promptly ineffective.
Summary of the invention: the problem of solution:
The present invention be directed to above-mentioned situation, be material screening with strong anti-wheat breed Brock and seek new and stable existence and closely linked molecule marker of powdery mildew resistance gene and method thereof with molecular biology method, be used for the wheat powdery mildew assisted selection; Because the research material therefor is the wheat breed (No. 15 physiological strains are the mixed type physiological strain, are the multiple Powdery Mildew germ of North China's popular) to No. 15 physiological strain immunity, it has the resistance of comparison broad-spectrum to North China white powder germ.Therefore, be expected to be cloned into new powdery mildew resistance gene according to the gained molecule marker.Technical scheme:
The molecule marker linked with powdery mildew resistance gene in wheat is characterized in that described molecule marker OPP15
900And Xgwm114
120, it obtains with following method:
(1) the disease-resistant gene donor parents is from Britain common wheat kind Brock, it is hybridized with susceptible common wheat parent 015 and susceptible parent capital 411, cross combination is " Brock/015//capital 411 ", disease-resistant individual plant is by hybridization, backcrosses and selfing, obtains from the stable advanced lines strain of mildew-resistance is;
(2) extract wheat parent seedling and filial generation seedling DNA with phenol-chloroform method;
(3) adopt RAPD and two kinds of molecule marking methods of SSR to carry out the screening of wheat powdery mildew resistance molecule marker;
(4) filter out a RAPD molecule marker OPP15
900With a SSR mark Xgwm114
120, identify through linksystem, find that these two marks and powdery mildew resistance gene in wheat are linked.
Adopt RAPD and SSR molecule marker to screen to be with the linked molecular marker method of powdery mildew resistance gene in wheat:
(1) RAPD, SSR primer are resisting, are feeling dna polymorphism analysis between parent and their filial generation:
Screen with the RAPD labeling technique, the 10bp oligonucleotide that adopts the exploitation of U.S. OPERON company is as random primer, and in the enterprising performing PCR amplification of PE-9700PCR instrument, the PCR reaction system is: wheat cdna group DNA (20ng/ μ l) 1 μ l, 10 * PCR Buffer, 2.5 μ l, MgCl
2(25mM) 2.5 μ l, dNTP (10mM) 0.5 μ l, primer (50 μ M) 0.5 μ l, Taq archaeal dna polymerase (5U/ μ 1) 0.25 μ l, ddH
2O 17.75 μ l, total system 25 μ l.Response procedures: 96 ℃ of sex change 1 minute, 35 ℃ of annealing 1 minute, 72 ℃ were extended 4 circulations 2 minutes; 94 ℃ of sex change 45 seconds, 36 ℃ of annealing 1 minute, 72 ℃ were extended 45 circulations 90 seconds; 72 ℃ of polishings 10 minutes; Product detects: containing 1.4% the agarose gel electrophoresis of 0.5% μ g/ μ l EB, ultraviolet lamp is observed down and the photographic recording result;
Screen with the SSR labeling technique, the SSR primer sequence synthetic primer that employing is delivered in 1998 according to R der is in the enterprising performing PCR amplification of PE-9700PCR instrument, PCR reaction system: 20ng/ μ 1 wheat cdna group DNA2.5 μ l, 10 * PCR Buffer, 2 μ l, 25mM MgCl
21.5 μ l, 2mM dNTP2 μ l, 20ng/ μ l primer 2 μ l, 5U/ μ l Taq archaeal dna polymerase 0.3 μ l, ddH
2O 9.7 μ l, total system 20 μ l; Response procedures: 94 ℃ of sex change 3 minutes, 94 ℃ of sex change 1 minute, 50 ℃ of (or 55 ℃, 60 ℃) annealing 1 minute, 72 ℃ were extended 2 minutes, 45 circulations, 72 ℃ of polishings 10 minutes; Product detects: (Acr: Bis=39: 1) electrophoresis, electrode buffer are 1 * TBE to 8% non-denaturing polyacrylamide gel, constant voltage 90-140 volt.Gel-colored employing cma staining; Experimental result record: take a picture and Tianjin, island CS-930 TLC-scanner record experimental result;
(2) linksystem of molecule marker is identified
Wheat hybridizing offspring powder mildew resistance is identified and is carried out in the field, powdery mildew is seeded in the stem and leaf of Wheat in boot stage, and the susceptible contrast strain of carrying disease germs is transplanted by the material to be identified of field, inoculates by natural propagation, 2-3 week " Invest, Then Investigate " disease resistance, the investigation rank is divided into immunity, susceptible two-stage; Extract overall dna disease-resistant, susceptible individual plant respectively, with carrying out the pcr amplification of individual plant with same reaction system, primer and the program in front, statistics resists, feels the frequency of occurrences and the switch type of individual plant indicia band and calculates crossover value, with the genetic distance between MapmakerEXP3.0b calculating mark and the mildew-resistance gene.Beneficial effect:
Powdery Mildew is a kind of serious plant disease that influences Wheat Production.The present invention utilizes molecule marking method to obtain two new and the closely linked molecule markers of powdery mildew resistance gene.Utilize this method, not only overcome shortcomings such as the conventional breeding method required time cycle is long, targetedly disease-resistant gene selected to obtain in the laboratory, also on purpose a plurality of mildew-resistance genes of polymerization, cultivate new variety of wheat with stable resistance, simultaneously also can utilize the molecular marker clone mildew-resistance gene of these two new powdery mildew resistance genes and it is carried out the 26S Proteasome Structure and Function analysis, this molecular genetics mechanism for further understanding wheat anti-powdery mildew has positive effect.Therefore, this result of study is all very valuable in wheat breeding practice and disease-resistant theoretical investigation.Its advantage specifically is summarized as follows:
(1) the of the present invention and closely linked molecule marker of powdery mildew resistance gene, it is the new mark that in the filial generation individual plant stable, obtains to the wheat breed Brock of North China's Powdery Mildew bacterial immunity and resistance thereof, No. 15 physiological strains all there is resistance, and stable existence, can be used for wheat powdery mildew cultivar identification and disease-resistant wheat offspring's assisted selection.
(2) this research material therefor is the wheat breed to No. 15 physiological strains (the mixed type physiological strain is the multiple Powdery Mildew germ of North China's popular) immunity, North China white powder germ is had the resistance of comparison broad-spectrum.Therefore, be expected to be cloned into new powdery mildew resistance gene according to the gained molecule marker.
(3) established good basis for clone's powdery mildew resistance gene in wheat, gene sequencing and the research of commentaries on classics mildew-resistance gene wheat.
Description of drawings: Fig. 1: the RAPD molecular marker screening electrophoretic band figure of four wheat breed genomic dnas; Fig. 2: detect OPP15
900With disease-resistant sex-linked electrophoretogram; Fig. 3: the SSR molecular marker screening electrophoretic band figure of four wheat breed genomic dnas; Fig. 4: detect Xgwm114
120With disease-resistant sex-linked electrophoretogram; Symbol is shown respectively not to be among Fig. 1-4: 1: susceptible parent capital 411; 2: susceptible common wheat parent 015; 3: disease-resistant gene donor parents Britain common wheat kind Brock; 4: the disease-resistant individual plant of filial generation " Brock/015//capital 411 ", disease-resistant individual plant are by hybridization, backcross and selfing, obtain from the stable advanced lines strain of mildew-resistance is; M: λ DNA Hind III+EcoR I Markers; R: the disease-resistant individual plant of filial generation that special band is arranged; R
*: do not have special band, but phenotype is disease-resistant filial generation individual plant; S: the susceptible individual plant of filial generation that does not have special band; S
*: special band is arranged, but phenotype is responsive filial generation individual plant;
Embodiment: (specifying in conjunction with the accompanying drawings) embodiment 1: use the RAPD molecule marking method, obtain the recruit mark OPP15 linked with powdery mildew resistance gene in wheat
900
Specific practice is: the disease-resistant gene donor parents is from Britain common wheat kind Brock, it is hybridized with susceptible common wheat parent 015 and susceptible parent capital 411, cross combination is " Brock/015//capital 411 ", disease-resistant individual plant is by hybridization, backcrosses and selfing, obtains 92 strain BC from the stable advanced lines strain of mildew-resistance is
2F
5One, extract DNA:
Get wheat leaf blade 0.2g, grind into powder in liquid nitrogen adds DNA extraction damping fluid (50mMTris-HCl, pH8.0 rapidly; 20mM EDTA, pH8.0; 50mM NaCl), 65 ℃ of temperature were bathed 15 minutes, put upside down mixing 2-3 time therebetween, 0 ℃ of ice bath 10 minutes.3000 rev/mins centrifugal 10 minutes, get supernatant liquor, add the saturated phenol of equal-volume Tris-HCl (pH8.0), put upside down mixing, 3000 rev/mins centrifugal 10 minutes.Get supernatant liquor, add equal-volume phenol, chloroform and primary isoamyl alcohol (25: 24: 1) extracting, put upside down mixing, 3000 rev/mins centrifugal 10 minutes.Get supernatant liquor, add the equal-volume chloroform, put upside down mixing, 3000 rev/mins centrifugal 10 minutes.Get supernatant liquor, add the cold ethanol of two volumes ,-20 ℃ of precipitations 2 hours, 12000 rev/mins centrifugal 20 minutes, the supernatant liquor that inclines, 70% washing with alcohol precipitation twice is dissolved in behind the natural airing among the 100 μ l TE.Add 1 μ l RNase (10mg/ml), 37 ℃ are incubated 1 hour, to remove the RNA in the sample, add isopyknic phenol, chloroform extracting once, 3000 rev/mins centrifugal 10 minutes, get supernatant liquor, use isopyknic chloroform extracting more once, the cold ethanol that adds two volumes,-20 ℃ of precipitations are more than 2 hours, 12000 rev/mins centrifugal 20 minutes, twice of 70% washing with alcohol precipitation, after air-dry, be dissolved among the 50 μ l TE.Ultraviolet spectrophotometry detects the concentration of DNA sample, and 0.7% agarose gel electrophoresis detects the integrity of DNA.Two, randomly amplified polymorphic DNA (RAPD) is analyzed:
As random primer (totally 20 of OPP01-OPP20,10mer), a part is given birth to worker bio-engineering corporation (totally 75 of 10mer) available from Shanghai to a RAPD primer part available from the 10bp oligonucleotide of U.S. OPERON company exploitation.1, pcr amplification: (1) reaction system is:
Wheat cdna group 20ng/ μ lDNA 1 μ l, 10 * PCR Buffer, 2.5 μ l, 25mM MgCl
22.5 μ l, 10mM dNTP 0.5 μ l, 50 μ M primers, 0.5 μ l, 5U/ μ l Taq archaeal dna polymerase 0.25 μ l, ddH
2O 17.75 μ l, total system 25 μ l.Mixing, centrifugal collection of moment, the PE9700 of U.S. PE company reacts.(2) response procedures:
96 ℃ of sex change 1 minute, 35 ℃ of annealing 1 minute, 72 ℃ were extended 4 circulations 2 minutes; 94 ℃ of sex change 45 seconds, 36 ℃ of annealing 1 minute, 72 ℃ were extended 45 circulations 90 seconds; 72 ℃ of polishings 10 minutes.2, electrophoresis detection:
Get amplified production 8 μ l, 1.4% sepharose (containing 0.5% μ g/ μ l EB) electrophoresis, ultraviolet lamp is observed down and is taken a picture.
We utilize 95 random primers that the RAPD analysis has been carried out in capital 411 and the disease-resistant individual plant of filial generation, finding wherein has 85 primers can amplify bands of a spectrum clearly, the amplified fragments size distribution is between 0.2kb-3.5kb, the amplified band number of each primer is the 1-10 bar, 5 of average out to are approximately analyzed genomic 420 seats.The result has only in the amplified production of primer OPP15 in Beijing 411 and the disease-resistant individual plant and shows polymorphism.Fig. 1 is the amplification of primer OPP15, can observe the special band OPP15 that a molecular weight is about 900bp in disease-resistant individual plant
900, repeated experiments three times, this band still can be stablized appearance.Continue to adopt OPP15 amplified hybridization parent Brock and 015 to find, can amplify special band OPP15 among the disease-resistant parent Brock
900, as shown in Figure 1: all can amplify special band OPP15 in 3 (disease-resistant parent Brock) and 4 (the disease-resistant individual plants of filial generation)
900, and lack OPP15 among 1 (susceptible parent capital 411) and 2 (the susceptible parents 015)
900Illustrating that this special band may exist with the wheat anti-powdery mildew characteristic get in touch.Three, RAPD molecule marker OPP15
900Linksystem identify:
Wheat hybridizing offspring powder mildew resistance is identified and is carried out in the field, powdery mildew is seeded in the stem and leaf of Wheat in boot stage, and the susceptible contrast strain of carrying disease germs is transplanted by the material to be identified of field, inoculates by natural propagation, 2-3 week " Invest, Then Investigate " disease resistance, the investigation rank is divided into immunity, susceptible two-stage.Extract overall dna disease-resistant, susceptible individual plant respectively, with above-mentioned same reaction system and program, carry out the pcr amplification of individual plant with primer OPP15, statistics resists, feels the frequency of occurrences and the switch type of individual plant indicia band and calculates crossover value, with the genetic distance between MapmakerEXP3.0b calculating mark and the mildew-resistance gene.With OPP15 92 individual plants of filial generation colony are carried out pcr amplification.The result shows that have 72 strains to have special band in 80 disease-resistant individual plants, 8 strains do not have special band; 5 strains have special band in 12 susceptible individual plants, and 7 strains do not have special band.This experimental result shows: specific DNA fragment OPP15
900With the disease resistance of wheat be closely linked, but in disease-resistant and susceptible individual plant, certain exchange is arranged all.R has OPP15 as shown in Figure 2
900The disease-resistant individual plant of the filial generation of special band; R
*Be not have OPP15
900Special band, but phenotype is disease-resistant filial generation individual plant, shows exchange has taken place; S does not have OPP15
900The susceptible individual plant of the filial generation of special band; S
*Be that OPP15 is arranged
900Special band, but phenotype is responsive filial generation individual plant, shows exchange has taken place.As calculated, its recombination fraction is 14.13%, OPP15
900And the genetic distance between disease-resistant gene is 16.6cM.
Embodiment 2: use the SSR molecule marking method, obtain the recruit mark Xgwm114 linked with powdery mildew resistance gene in wheat
120
Vegetable material is with embodiment 1, and specific practice is as follows: one, extract DNA:
Respectively get wheat leaf blade 0.2g, grind into powder in liquid nitrogen adds DNA extraction damping fluid (50mMTris-HCl, pH8.0 rapidly; 20mM EDTA, pH8.0; 50mM NaCl, 65 ℃ of temperature were bathed 15 minutes, put upside down mixing 2-3 time therebetween, 0 ℃ of ice bath 10 minutes.3000 rev/mins centrifugal 10 minutes, get supernatant liquor, add the saturated phenol of equal-volume Tris-HCl (pH8.0), put upside down mixing, 3000 rev/mins centrifugal 10 minutes.Get supernatant liquor, add equal-volume phenol, chloroform and primary isoamyl alcohol (25: 24: 1) extracting, put upside down mixing, 3000 rev/mins centrifugal 10 minutes.Get supernatant liquor, add the equal-volume chloroform, put upside down mixing, 3000 rev/mins centrifugal 10 minutes.Get supernatant liquor, add the cold ethanol of two volumes ,-20 ℃ of precipitations 2 hours, 12000 rev/mins centrifugal 20 minutes, the supernatant liquor that inclines, 70% washing with alcohol precipitation twice is dissolved in behind the natural airing among the 100 μ l TE.Add 1 μ l RNase (10mg/ml), 37 ℃ are incubated 1 hour, to remove the RNA in the sample, add isopyknic phenol, chloroform extracting once, 3000 rev/mins centrifugal 10 minutes, get supernatant liquor, use isopyknic chloroform extracting more once, the cold ethanol that adds two volumes,-20 ℃ of precipitations are more than 2 hours, 12000 rev/mins centrifugal 20 minutes, twice of 70% washing with alcohol precipitation, after air-dry, be dissolved among the 50 μ l TE.Ultraviolet spectrophotometry detects the concentration of DNA sample, and 0.7% agarose gel electrophoresis detects the integrity of DNA.Two, the SSR primer is synthetic: the SSR primer sequence compound experiment institute that delivered in 1998 according to R der
Need primer (synthetic) by Shanghai bio-engineering corporation.Three, micro-satellite primers (SSR) is analyzed: 1, pcr amplification:
(1) reaction system: 20ng/ μ l wheat cdna group DNA 2.5 μ l, 10 * PCR Buffer, 2 μ l, 25mMMgCl
21.5 μ l, 2mM dNTP2 μ l, 20ng/ μ l primer 2 μ l, 5U/ μ l Taq archaeal dna polymerase 0.3 μ l, ddH
2O 9.7 μ l, total system 20 μ l.Mixing, centrifugal collection of moment is reacted on the U.S. PE-9700.(2) response procedures:
94 ℃ of sex change 3 minutes, 94 ℃ of sex change 1 minute, 50 ℃ of (or 55 ℃, 60 ℃) annealing 1 minute, 72 ℃ were extended 2 minutes, 45 circulations, 72 ℃ of polishings 10 minutes.2, electrophoresis detection: (1) gel electrophoresis:
Get amplified production 5 μ l, (Acr: Bis=39: 1) electrophoresis, electrode buffer are 1 * TBE to 8% non-denaturing polyacrylamide gel, constant voltage 90-140 volt.(2) gel-colored: 10% acetic acid is fixed, rinsed with deionized water 3 times, 0.1% Silver Nitrate 250ml (adding 40% formaldehyde, 375 μ l) dyeing, rinsed with deionized water 10 seconds, put into the developing solution (the 7.5g anhydrous sodium carbonate is dissolved in 250ml deionized water+40% formaldehyde, 375 μ l+10mg/ml Sulfothiorine, 50 μ l) of precooling immediately, it is clear constantly to shake to the DNA band, 10% acetic acid termination reaction.3, experimental result record:
Take a picture and Tianjin, island CS-930 TLC-scanner record experimental result.
Utilize 20 micro-satellite primers that ssr analysis has been carried out in capital 411 and filial generation resistant, susceptible individual plant, finding wherein has 17 primers can amplify bands of a spectrum clearly, the amplified fragments size distribution is between 110bp-300bp, the amplified band number of each primer is the 1-5 bar, average 4, approximately genomic 65 seats are analyzed.The result has only in the amplified production of primer Xgwm114 in Beijing 411 and the disease-resistant individual plant and shows polymorphism.Fig. 3 is the amplification of primer Xgwm114, can observe the special band Xgwm114 that a molecular weight is about 120bp in 3 (disease-resistant parents), 4 (disease-resistant filial generation individual plant)
120, repeated experiments three times, this band still can be stablized appearance, and does not have this band in 1 (susceptible parent capital 411) and 2 (susceptible parents 015).Illustrate that this special band may exist certain to get in touch with the wheat anti-powdery mildew characteristic.
The Xgwm114 primer sequence is: left wing 5 '-ACA AAC AGA AAA TCA AAA CCC G-3 ', and right flank 5 '-ATC CAT CGC CAT TGG AGT G-3 ' is positioned on wheat 3B and the 3D karyomit(e), and the renaturation temperature is 60 ℃.Four, SSR molecule marker Xgwm114
120Linksystem identify:
Adopt primer Xgwm114 to carry out the pcr amplification of individual plant, its method together
Embodiment 1Linksystem identify.
In order to detect Xgwm114
120Stability and with the linkage relationship of disease-resistant gene, with primer Xgwm114 92 individual plants of filial generation are increased, as shown in Figure 4.Have 63 strains (R) to have special band in 80 disease-resistant individual plants as a result, 17 strains do not have special band (R
*); 3 strain (S in 12 susceptible individual plants
*) have special band, 9 strains not to have special band (S).As calculated, its recombination fraction is 21.74%, Xgwm114
120And the genetic distance between disease-resistant gene is 28.5cM.
Claims (2)
1, with the linked molecule marker of powdery mildew resistance gene in wheat, it is characterized in that described molecule marker OPP15
900And Xgwm114
120, it obtains with following method:
(1) the disease-resistant gene donor parents is from Britain common wheat kind Brock, it is hybridized with susceptible common wheat parent 015 and susceptible parent capital 411, cross combination is " Brock/015//capital 411 ", disease-resistant individual plant is by hybridization, backcrosses and selfing, obtains from the stable advanced lines strain of mildew-resistance is;
(2) extract wheat parent seedling and filial generation seedling DNA with phenol-chloroform method;
(3) adopt RAPD and two kinds of molecule marking methods of SSR to carry out the screening of wheat powdery mildew resistance molecule marker;
(4) filter out a RAPD molecule marker OPP15
900With a SSR mark Xgwm114
120, identify through linksystem, find that these two marks and powdery mildew resistance gene in wheat are linked.
2, according to claim 1 described with the linked molecule marker of powdery mildew resistance gene in wheat, it is characterized in that adopting RAPD and SSR molecule marker to screen being with the linked molecular marker method of powdery mildew resistance gene in wheat:
(1) RAPD, SSR primer are resisting, are feeling dna polymorphism analysis between parent and their filial generation:
Screen with the RAPD labeling technique, the 10bp oligonucleotide that adopts the exploitation of U.S. OPERON company is as random primer, and in the enterprising performing PCR amplification of PE-9700PCR instrument, the PCR reaction system is: 20ng/ μ l wheat cdna group DNA1 μ l, 10 * PCR Buffer, 2.5 μ l, 25mM MgCl
22.5 μ l, 10mMdNTP0.5 μ l, 50 μ M primers, 0.5 μ l, 5U/ μ l Taq archaeal dna polymerase 0.25 μ l, ddH
2O 17.75 μ l, total system 25 μ l.Response procedures: 96 ℃ of sex change 1 minute, 35 ℃ of annealing 1 minute, 72 ℃ were extended 4 circulations 2 minutes; 94 ℃ of sex change 45 seconds, 36 ℃ of annealing 1 minute, 72 ℃ were extended 45 circulations 90 seconds; 72 ℃ of polishings 10 minutes; Product detects: containing 1.4% the agarose gel electrophoresis of 0.5% μ g/ μ l EB, ultraviolet lamp is observed down and the photographic recording result;
Screen with the SSR labeling technique, the SSR primer sequence synthetic primer that employing is delivered in 1998 according to R der is in the enterprising performing PCR amplification of PE-9700PCR instrument, PCR reaction system: 20ng/ μ l wheat cdna group DNA2.5 μ l, 10 * PCR Buffer, 2 μ l, 25mM MgCl
21.5 μ l, 2mM dNTP2 μ l, 20ng/ μ l primer 2 μ l, 5U/ μ l Taq archaeal dna polymerase 0.3 μ l, ddH
2O 9.7 μ l, total system 20 μ l; Response procedures: 94 ℃ of sex change 3 minutes, 94 ℃ of sex change 1 minute, 50 ℃ of (or 55 ℃, 60 ℃) annealing 1 minute, 72 ℃ were extended 2 minutes, 45 circulations, 72 ℃ of polishings 10 minutes; Product detects: (Acr: Bis=39: 1) electrophoresis, electrode buffer are 1 * TBE to 8% non-denaturing polyacrylamide gel, constant voltage 90-140 volt.Gel-colored employing cma staining; Experimental result record: take a picture and Tianjin, island CS-930 TLC-scanner record experimental result; (2) linksystem of molecule marker is identified:
Wheat hybridizing offspring powder mildew resistance is identified and is carried out in the field, powdery mildew is seeded in the stem and leaf of Wheat in boot stage, and the susceptible contrast strain of carrying disease germs is transplanted by the material to be identified of field, inoculates by natural propagation, 2-3 week " Invest, Then Investigate " disease resistance, the investigation rank is divided into immunity, susceptible two-stage; Extract overall dna disease-resistant, susceptible individual plant respectively, with carrying out the pcr amplification of individual plant with same reaction system, primer and the program in front, statistics resists, feels the frequency of occurrences and the switch type of individual plant indicia band and calculates crossover value, with the genetic distance between MapmakerEXP3.0b calculating mark and the mildew-resistance gene.
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| CN1322133C (en) * | 2005-07-12 | 2007-06-20 | 南京农业大学 | Wheat anti-powdery mildew gene Pm21 linked codorminant PCR marker and its usage |
| CN1328389C (en) * | 2004-12-30 | 2007-07-25 | 南京农业大学 | Tagged primer chained to Pm21 gene of anti powdery mildew of wheat and application |
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2002
- 2002-09-27 CN CN 02131314 patent/CN1252284C/en not_active Expired - Fee Related
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| CN100336905C (en) * | 2004-05-08 | 2007-09-12 | 南京农业大学 | Wheat fertility recovery gene molecular mark and its obtaining method |
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| CN112176083B (en) * | 2019-07-05 | 2022-04-12 | 中国科学院遗传与发育生物学研究所 | Functional molecular marker of wheat powdery mildew resistance related gene Pm41 and application thereof |
| CN111996283A (en) * | 2020-09-22 | 2020-11-27 | 烟台大学 | Molecular marker closely linked with wheat powdery mildew resistance gene PmLS5082 and application thereof |
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