CN100494401C - SCAR molecule marker closely linked with wheat anti powdery mildew gene - Google Patents
SCAR molecule marker closely linked with wheat anti powdery mildew gene Download PDFInfo
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Abstract
A molecular marker SCAR associated with the wheat gene resisting powdery mildew features that the RAPD molecular marking method is used to mark the resistances of wheat parents and their hybrid descendant 'Brock/015//Jing411' and screen from them. The 10bp oligonucleoside is used as random primer to obtain new molecular marker S2092 900, which is then transformed to stable SCAR markers: SCAR860 and SCAR200.
Description
Technical field
The invention belongs to agricultural biotechnology engineering, particularly with closely linked SCAR molecule marker of powdery mildew resistance gene and preparation method thereof.
Technical background
The wheat powdery mildew that is caused by obligatory parasitism fungi wheat powdery mildew (Erysiphe graminisf.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 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 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.Just 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, but still has the unknown gene of a lot of mildew-resistances, does not filter out molecule marker as yet.Therefore, continue to use molecular marker method, screen the mark relevant, significant application value is arranged on agricultural with mildew-resistance gene.
Summary of the invention
The problem that solves:
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, thereby overcome shortcomings such as conventional genetic breeding cycle is long; 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 SCAR molecule marker linked with powdery mildew resistance gene in wheat is characterized in that described molecule marker SCAR
860, SCAR
200, it is to filter out S2092 with the RAPD molecule marking method
972Molecule marker is again with S2092
972Convert stable SCAR mark to, this mark adopts following method to obtain:
(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 the RAPD molecule marking method, carry out the screening of the molecule marker relevant with the wheat powdery mildew resistance;
(4) filter out a RAPD molecule marker S2092
900, identify through linksystem, find that this mark and powdery mildew resistance gene in wheat are linked, with the genetic distance of resistant gene be 4.3cM;
(5) with S2092
900Clone and order-checking also are converted to SCAR mark, i.e. SCAR
860, SCAR
200
Adopt the RAPD molecule marking method, carry out the molecule marker linked with powdery mildew resistance gene in wheat, the specific practice of its screening is:
Label screening: utilize 120 Operon and 93 S series random primers (10bp) between resistance parent Brock, sense parent capital 411 and their hybridization, backcross progeny resistance individual plant, to carry out the dna polymorphism analysis, promptly with the screening of RAPD labeling technique, adopt the 10bp oligonucleotide as random primer, 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, 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, response procedures: 1 minute, 35 ℃ annealing of 96 ℃ of sex change are extended 2 minutes, 4 circulations for 1 minute, 72 ℃, and 45 seconds, 36 ℃ annealing of 94 ℃ of sex change are extended 90 seconds, 45 circulations for 1 minute, 72 ℃, 72 ℃ of polishings 10 minutes; Product detects: containing 1.0% the agarose gel electrophoresis of 0.5% μ g/ μ l EB, ultraviolet lamp is observed down and the photographic recording result.
The linksystem of molecule marker is identified: utilize Brock * capital 411F
2Colony's 131 strain individual plants are that material carries out linksystem analysis between molecule marker and disease-resistant gene.Wheat hybridizing offspring (131 strain individual plant) 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 divides 0,0; Be immunity, the 1-4 level is represented high-strength anti-, anti-, responsive, extremely sensitive respectively.Resistance is identified and is shown, 101 strains are disease-resistant, 30 strains are susceptible, 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 S2092, statistics is anti-, the frequency of occurrences and the switch type of sense individual plant indicia band and calculate crossover value, calculates genetic distance between mark and the mildew-resistance gene with MapmakerEXP3.0b.Critical LOD value is 3.0, according to the Kosambi function,
Recombination fraction is transformed into map unit centimorgan that (cM).
The conversion of SCAR mark: at first with S2092
900Be cloned in bacterial plasmid
On-T easy the Vector, after the both-end order-checking, S2092
900The physical length of molecule marker is that (submit GenBank to, accession number is 972bp: AY324385), also synthesize 3 SCAR primers, called after S2092 according to this sequences Design
972A, S2092
972B, S2092
972C, its A and B are that a pair of, A and C are a pair of, at Brock, the (Brock/015//capital 411 of hybridizing, backcross
7) increase respectively in offspring's resistance individual plant, in the resistance individual plant, all can amplify two SCAR mark SCAR of expectation
860And SCAR
200. in order to detect the reliability of these two SCAR marks, we are to the F in Brock * capital 411
2The anti-sense of segregating population individuality is analyzed.The result shows, at F
2All detected this two SCAR marks in the isolated resistance individuality, and not do not found in the susceptible individual, the rule that SCAR is marked at is anti-, occur in the sense individual plant is consistent with the RAPD mark.
The specific practice of above-mentioned transformation is as follows:
A) the segmental cloning and sequencing of resistance
With reference to the classical segmental experimental technique low melting-point agarose of cloned DNA purified pcr product,
-Teasy Vector (Promega) connects, transformed into escherichia coli Jm109, and recombinant plasmid is through T7, Sp6 PCR electrophoresis detection, and EcoR I enzyme is cut evaluation, obtains positive colony, send Sangon order-checking.Sequencing result is analyzed with information biology SMS, and carried out homology analysis by Internet and the international gene database of GenBank.
B) design of primers and SCAR analyze
With according to S2092
972The above-mentioned two pairs of SCAR primers of sequences Design and synthetic
S2092
972A 5’GGTTGTTCCC?AAAGAGGTCA?3’
S2092
972B 5’ATGTTTGAAGC?AGGGTGCAG?3’
S2092
972C 5’TCCTCAATCTGGAGGGACAC?3’，
At Brock, the (Brock/015//capital 411 of hybridizing, backcross
7) increase respectively in offspring's resistance individual plant, reaction conditions is as follows: reaction system is 25 μ l, and the content of each component materials is respectively template 20ng; 10 * Buffer, 2.5 μ l; MgCl
21.6mM; DNTP 0.15mmol/L; 5pmol primer; Taq 1.25U, ddH
2O polishing 25 μ l, mixing, centrifugal collection is reacted on PTC-150-25, and response procedures is as follows: 94 ℃ of sex change 3min, 64 ℃ of annealing 2min, 72 ℃ are extended 2min, 30 circulations, 72 ℃ of 5min, 4 ℃ of preservations.1.0% sepharose (containing EB), electrophoresis 1 hour, ultraviolet reflectance tem analysis instrument are observed and are taken a picture.
Beneficial effect:
Powdery Mildew is a kind of serious plant disease that influences Wheat Production.The present invention utilizes molecule marking method to obtain new and the closely linked RAPD molecule marker of powdery mildew resistance gene, and by clone, order-checking, design of primers, again the RAPD mark is converted to stable SCAR mark.Utilize this method, shortcomings such as conventional breeding method required time cycle length have not only been overcome, and can targetedly disease-resistant gene be selected to obtain in the laboratory, on purpose a plurality of mildew-resistance genes of polymerization, cultivate new variety of wheat with stable resistance, also can utilize simultaneously the molecular marker clone mildew-resistance gene of this new powdery mildew resistance gene, 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 (be the mixed type physiological strain, be 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) originally be labeled as stable SCAR mark, and with mildew-resistance gene close linkage (genetic distance is 4.3cM), can be directly used in the assisted selection of wheat anti-powdery mildew molecule marker, establish good basis for clone's powdery mildew resistance gene in wheat, gene sequencing and the research of commentaries on classics mildew-resistance gene wheat simultaneously.
Description of drawings
Fig. 1: the RAPD molecule marker S2092 of capital 411, resistance individual plant, Brock genomic dna
900Screening electrophoretic band figure;
Fig. 2: detection molecules mark S2092
900The electrophoretic band figure chain with mildew-resistance;
Fig. 3: SCAR
860, SCAR
200The mark band diagram;
Fig. 4: SCAR
860And SCAR
200Be marked at the distribution band diagram in disease-resistant parent and the disease-resistant individual plant;
Symbol is expressed as respectively among Fig. 1-4:
1: susceptible parent capital 411; 2: the resistance individual plant; 3: disease-resistant variety Brock;
M：λDNA?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;
Embodiment: (specifying in conjunction with the accompanying drawings)
Embodiment 1:
Use the RAPD molecule marking method, obtain the molecule marker S2092 linked with powdery mildew resistance gene in wheat
900. the physical length of this molecule marker is 972bp, converts SCAR to the SCAR method
860, SCAR
200Two stable molecule markers.
Specific practice is: the disease-resistant gene donor parents is Britain common wheat kind Brock, and it is hybridized with susceptible common wheat strain 015 and susceptible parent capital 411, backcrosses with capital 411, is combined as " Brock/015//capital 411
7", disease-resistant individual plant is by hybridization, backcrosses and selfing, obtains from the stable advanced lines strain of mildew-resistance is.Anti-, the sense individual plant that are used for the linksystem evaluation then are Brock/ capital 411F
2Obtain middle the separation, comprises individual and 30 susceptible individuals of 101 resistances;
One, extracts 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, amplification polymorphism DNA (RAPD) analyzes:
The RAPD primer is 120 of OP series, available from 93 of the S of U.S. OPERON company (being the 10bp oligonucleotide) series, and available from Shanghai bio-engineering corporation, 213 altogether.
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 213 random primers that the RAPD analysis has been carried out in capital 411 and the disease-resistant individual plant of filial generation, find to have only among the amplified production in Beijing 411 of primer S2092 and disease-resistant individual plant and the Brock to show polymorphism.Fig. 1 is the amplification of primer S2092, can observe the special band S2092 that a molecular weight is about 900bp in disease-resistant individual plant and Brock
900, repeated experiments three times, this band still can be stablized appearance.As shown in Figure 1: 1, capital 411,2, disease-resistant individual plant, 3, Brock.Illustrating that this special band may exist with the wheat anti-powdery mildew characteristic get in touch.
Three, RAPD molecule marker S2092
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 the " Invest, Then Investigate " disease resistance, the investigation rank is divided into immunity, strong anti-, anti-, sense and high sense.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 S22092, 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 S2092 131 individual plants of filial generation colony are carried out pcr amplification.The result shows that have 95 strains to have special band in 101 disease-resistant individual plants, 6 strains do not have special band; All there is not special band in 30 susceptible individual plants.This experimental result shows: specific DNA fragment S2092
900With the disease resistance of wheat be closely linked, but in disease-resistant and susceptible individual plant, certain exchange is arranged all.R has S2092 as shown in Figure 2
900The disease-resistant individual plant of the filial generation of special band; R
*Be not have S2092
900Special band, but phenotype is disease-resistant filial generation individual plant, shows exchange has taken place; S does not have S2092
900The susceptible individual plant of the filial generation of special band; As calculated, S2092
900And the genetic distance between disease-resistant gene is 4.3cM.
Four, S2092
900Clone, order-checking and the conversion of SCAR mark
For with S2092
900Convert stable SCAR mark to, with S2092
900Dna fragmentation low melting-point agarose gel-purified, the dna fragmentation of purifying with
-T easy Vector (Promega) connects, transformed into escherichia coli Jm109, and recombinant plasmid is through T7, Sp6 PCR electrophoresis detection, and EcoR I enzyme is cut evaluation, obtains positive colony, send Sangon order-checking.Sequencing result is analyzed with information biology SMS, and carried out homology analysis by Internet and the international gene database of GenBank.After the both-end order-checking, S2092
900Mark is made up of 972 bp altogether.Owing to do not inquire relevant gene order and this fragment homology from GenBank, this fragment can be thought new molecule marker linked with mildew-resistance gene among the Brock.
According to the dna sequence dna of cloned sequence, we have synthesized the SCAR labeled primer of 3 20 bases, and called after S2092
972A, S2092
972B, S2092
972C, sequence is as follows:
S2092
972A?5’GGTTGTTCCC?AAAGAGGTCA?3’
S2092
972B?5’ATGTTTGAAGC?AGGGTGCAG?3’
S2092
972C?5’TCCTCAATCTGGAGGGACAC?3’
Wherein primer A contains the 10mer primer sequence of RAPD molecule marker S2092, and extends 10 bases to 3 ' end, and primer B and C are respectively according to S2092
972Base designs in the sequence, does not contain the random primer sequence of RAPD molecule marker S2092.With above-mentioned primer at Brock, the (Brock/015//capital 411 of hybridizing, backcross
7) increase respectively in offspring's resistance individual plant, reaction conditions is: reaction system is 25 μ l, and the content of each component materials is respectively template 20ng; 10 * Buffer, 2.5 μ l; MgCl
21.6mM; DNTP0.15mmol/L; 5pmol primer; Taq 1.25U, ddH
2O polishing 25 μ l, mixing, centrifugal collection is reacted on PTC-150-25, and response procedures is as follows: 94 ℃ of sex change 3min, 64 ℃ of annealing 2min, 72 ℃ are extended 2min, 30 circulations, 72 ℃ of 5min, 4 ℃ of preservations.1.0% sepharose (containing EB), electrophoresis 1 hour, ultraviolet reflectance tem analysis instrument are observed and are taken a picture.Amplification proves, at Brock, the (Brock/015//capital 411 of hybridizing, backcross
7) amplify 860bp and two special bands of 200bp respectively in offspring's resistance individual plant.Then do not detect these two special bands in Beijing 411 as shown in Figure 3.In Fig. 3, M1:DL2000Marker, M2: λ DNA EcoRI/HindIII Marker.1,2:SCAR
8603,4:SCAR
200.
In order to detect the reliability of these two SCAR marks, we are to the F in Brock * capital 411
2The anti-sense of segregating population individuality is analyzed.The result shows, at F
2All detected this two SCAR marks in the isolated resistance individuality, and do not found in the susceptible individual, as shown in Figure 4.In Fig. 4, M is λ DNAEcoRI/HindIII Marker.
Claims (4)
1. the mutually closely linked molecule marker of powdery mildew resistance gene in wheat is characterized in that described molecule marker SCAR
860, SCAR
200, be to filter out S2092 with the RAPD molecule marking method
972Molecule marker is again with S2092
972Convert stable SCAR mark to, this mark adopts following method to obtain:
(1) the disease-resistant gene donor parents is from Britain common wheat kind Brock, it is hybridized with susceptible common wheat strain 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 the RAPD molecule marking method, carry out the screening of the molecule marker relevant with the wheat powdery mildew resistance;
(4) filter out a RAPD molecule marker S2092
900, identify through linksystem, find that this mark and powdery mildew resistance gene in wheat are linked, with the genetic distance of resistant gene be 4.3cM;
(5) with S2092
900Be cloned in bacterial plasmid
On the easy Vector, after the both-end order-checking, S2092
900The physical length of molecule marker is 972bp, and this is marked at all can amplify SCAR mark SCAR in the resistance individual plant
860And SCAR
200
According to claim 1 described with the closely linked molecule marker of powdery mildew resistance gene in wheat, it is characterized in that adopting the RAPD molecule marking method screening molecule marker relevant with the wheat powdery mildew resistance, specific practice is: resisting with the 10bp random primer, dna polymorphism analysis between sense parent and their filial generation, promptly with the screening of RAPD labeling technique, the 10bp oligonucleotide that adopts the exploitation of U.S. OPERON company is as random primer, 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, 10mMdNTP 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, response procedures: 1 minute, 35 ℃ annealing of 96 ℃ of sex change are extended 2 minutes, 4 circulations for 1 minute, 72 ℃, and 45 seconds, 36 ℃ annealing of 94 ℃ of sex change are extended 90 seconds, 45 circulations for 1 minute, 72 ℃, 72 ℃ of polishings 10 minutes; Product detects: containing 1.0% the agarose gel electrophoresis of 0.5% μ g/ μ l EB, ultraviolet lamp is observed down and the photographic recording result.
According to claim 1 described with the closely linked molecule marker of powdery mildew resistance gene in wheat, the linksystem that it is characterized in that molecule marker is identified, be that the evaluation of wheat hybridizing offspring powder mildew resistance is carried out in the field, powdery mildew is seeded in the stem and leaf of Wheat in boot stage, the susceptible contrast strain of carrying disease germs is transplanted by the material to be identified of field, inoculate by natural propagation, 2-3 week " Invest, Then Investigate " disease resistance, the investigation rank is divided into immunity, strong anti-, anti-, sense and high sense, extract disease-resistant respectively, the overall dna of susceptible individual plant, with PCR reaction system and the program in the label screening, be that 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, 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, response procedures: 1 minute, 35 ℃ annealing of 96 ℃ of sex change are extended 2 minutes, 4 circulations for 1 minute, 72 ℃, and 45 seconds, 36 ℃ annealing of 94 ℃ of sex change are extended 90 seconds, 45 circulations for 1 minute, 72 ℃, 72 ℃ of polishings 10 minutes; Carry out the pcr amplification of individual plant with primer S2092, statistics is anti-, the frequency of occurrences and the switch type of sense individual plant indicia band and calculate crossover value, calculates genetic distance between mark and the mildew-resistance gene with MapmakerEXP3.0b.
4. the described and closely linked molecule marker of powdery mildew resistance gene in wheat according to claim 1 is characterized in that S2092
900Convert stable SCAR mark to, be achieved in that at first this marker clone at bacterial plasmid
On the easy Vector, after the both-end order-checking, S2092
900The physical length of molecule marker is 972bp, according to this sequences Design and synthetic 3 SCAR primers, called after S2092
-900A, S2092
-900B, S2092
-900C, its A and B are that a pair of, A and C are a pair of, in Brock hybridization, to backcross be Brock/015//capital 411
7Increase respectively in offspring's resistance individual plant, in the resistance individual plant, all can amplify two SCAR mark SCAR of expectation
860And SCAR
2003 SCAR primers are promptly:
S2092
972A 5’GGTTGTTCCC?AAAGAGGTCA3’；
S2092
972B 5’ATGTTTGAAGC?AGGGTGCAG3’；
S2092
972C 5’TCCTCAATCTGGAGGGACAC3’。
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| CNB2003101220621A CN100494401C (en) | 2003-12-31 | 2003-12-31 | SCAR molecule marker closely linked with wheat anti powdery mildew gene |
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| CN100494401C true CN100494401C (en) | 2009-06-03 |
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| 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 |
| 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 |
| CN100434533C (en) * | 2006-05-23 | 2008-11-19 | 天津师范大学 | Molecular marking closely linked with wheat Brock powdery mildew resistant gene |
| CN101565751B (en) * | 2009-06-01 | 2012-02-08 | 四川农业大学 | Development of wide stress protein family gene specific molecular marker and application thereof |
| CN102533745B (en) * | 2012-01-10 | 2013-03-06 | 山东省林业科学研究院 | Saline-alkaline-tolerance Fraxinus velutina sequence-characterized amplified region (SCAR) marker and applications in assistant selection breeding |
| CN102517398B (en) * | 2012-01-10 | 2013-05-15 | 山东省林业科学研究院 | Molecular detection method for rapidly verifying salt-tolerant fraxinus velutina |
| CN103243164A (en) * | 2013-05-06 | 2013-08-14 | 西北农林科技大学 | Method for identifying psathyrostachys huashanica allogenic material in wheat genome |
| CN104313021B (en) * | 2014-10-21 | 2017-01-18 | 山西省农业科学院作物科学研究所 | Molecular marker of wheat powdery mildew disease-resistant genes Pm51 and application of molecular marker |
| CN106222285B (en) * | 2016-08-11 | 2019-06-18 | 江苏省农业科学院 | The molecular labeling isolated with powdery mildew resistance gene in wheat Pm48 |
| CN107338323B (en) * | 2017-09-08 | 2020-09-29 | 贵州省烟草科学研究院 | Molecular marker closely linked with powdery mildew resistance of flue-cured tobacco variety TT7 and application thereof |
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| 小麦抗白粉病基因的分子标记. 中国农业大学学报，第8卷第1期. 2003 |
| 小麦抗白粉病基因的分子标记. 中国农业大学学报，第8卷第1期. 2003 * |
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| 贵农21白粉病抗性基因的RAPD分子标记. 山地农业生物学报，第20卷第5期. 2001 * |
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