CN101570790B - Powdery mildew resistant wheat auxiliary screening method and special primer thereof - Google Patents
Powdery mildew resistant wheat auxiliary screening method and special primer thereof Download PDFInfo
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Abstract
The invention discloses a powdery mildew resistant wheat auxiliary screening method and a special primer thereof. The powdery mildew resistant wheat auxiliary screening primer pairs provided by the invention are at least one pair of the primer pairs of Xbarc79-1 and Xwmc288-1; the deoxyribonucleotide sequence of the primer pairs Xbarc79-1 is shown as sequence 1 and sequence 2 in a sequence table; the deoxyribonucleotide sequence of the primer pair Xwmc288-1 is shown as sequence 3 and sequence 4 in the sequence table. The invention also provides molecular markers Xbarc79 and Xwmc288 of powdery mildew resistant gene, the deoxyribonucleotide sequence of the molecular marker Xbarc79 is shown as sequence 5 in the sequence table, and the deoxyribonucleotide sequence of the molecular marker Xwmc288 is shown as sequence 6 in the sequence table. The primer pairs and the molecular marker of the invention can play an important role in wheat breeding for disease resistance.
Description
Technical field
The present invention relates to a kind of method and primer special thereof of powdery mildew resistant wheat auxiliary screening.
Background technology
Wheat powdery mildew is the fungal disease that is caused by wheat powdery mildew (Blumeria graminis f.sp.tritici), and the past mainly takes place in the area that has a moderate climate, humidity is rainy.The popularization of and semi-dwarf mutant kind of short stem and the continuous improvement of water and fertilizer condition along with wheat, the Wheat Production level increases substantially, the high-density that causes of the amount of broadcasting and irrational cultivation steps such as nitrogen fertilizer application, overirrigation greatly partially, the condition of bringing out of Powdery Mildew is provided artificially, the harm of Powdery Mildew is increased the weight of day by day.Before the seventies in 20th century, mainly moistening rainy southwest and Shandong coastal area are popular in Yunnan Province of China, Guizhou etc. for wheat powdery mildew, and should disease develop into one of main disease in northern winter wheat district after 80 generations.At present, Powdery Mildew has become one of main disease of China and world wheat, generally can make wheat yield 5～19% in the time that disease takes place, and the serious time underproduction is up to 30%.According to statistics, the generation on the wheat planting area in the whole nation 39% of nineteen ninety wheat powdery mildew is popular, and the production loss that causes reaches 14.39 hundred million kg.Seed selection anti-disease wheat kind is most economical, the safe and effective method of control wheat powdery mildew.The powdery mildew resistance gene in wheat of having named at present has 58, be distributed in 39 sites, except that Pm38 and Pm39 (becoming the strain disease-resistant gene), all the other named mildew-resistance genes mostly are the main disease-resistant gene of imitating of physiological specialization, because it shows as high resisting and be subjected to liking of breeding man deeply Powdery Mildew.Because the physiological strain complexity of wheat powdery mildew, variation is fast, fecundity is strong, therefore causes the main disease-resistant gene resistance of imitating to lose fast, variety resistance can not lasting stability, has very big hidden danger in the production.As the seventies in last century, the anti-source of the powdery mildew major part that used wheat breed is gone up in China's production to the mid-80, along with Pm8 gene resistance is lost in succession in China most of wheat planting district, causes wheat powdery mildew to be very popular in China from Pm8.Adult plant resistance is non-microspecies specialization, mainly be by becoming the strain phase to postpone infecting and breed and showing disease resistance of germ, reduced the selective pressure of wheat breed to the pathogenic bacteria physiological strain, the resistance time length is prolonged, the tremendous economic that helps avoiding the disease resistance forfeiture to cause is lost, therefore, become strain mildew-resistance kind to be subjected to the attention of Chinese scholars day by day.
Molecule marker (comprising RAPD, RFLP, SSR and RGAP) has obtained widespread use in the gene mapping of wheat, in recent years, 28 powdery mildew resistance gene in wheat have been located with molecule marker, wherein the SSR mark is because advantages such as its polymorphism height, codominance and good reproducibilities, be fit to the assignment of genes gene mapping and mapping, and the application of on some mildew-resistance genes, having succeeded of this method.For example, main disease-resistant gene Pm24, Pm27, Pm30, Pm5e, Pm33, MlZec1, Pm2, PmY39, Pm12, Pm35, Mlm3033 and Mlm80, the Pm37 of imitating, and become strain disease-resistant gene Pm38 and Pm39 all to obtain closely linked SSR mark, and be used for mark assisting sifting and pyramiding breeding.
Hundred farmings 64 are wheat breeds that Henan Province's authorization in 1998 is promoted, be that wheat breed is promoted mainly in geographic one of China the Yellow River and Huai He River sea, from 1993 to 2000, annual average cultivated area is 700,000 hectares, remain stable disease resistance for many years to all powdery mildew physiological strains, identify through (2005) such as Wang Zhulin, think that comparatively typically becomes a strain powdery-mildew-resistance wheat kind.This product grow wheat shows as high-quality, high yield and wide adaptability in the field, be widely used as the parent in the wheat breeding practice.At present, with hundred farmings 64 be parent and the wheat breed by authorization have in Luo 9908, the Huaihe River wheat 0320,04 36 and the source educate No. three, and Xu wheat 4060 that is in the Gao Daiqu examination with dredge wheat 35 etc., these wheat breeds all show as mildew-resistance in the field.This shows that important effect is being brought into play in hundred farmings 64 in China's wheat breeding.Wang Zhulin etc. (2005) carry out genetic analysis to hundred farmings 64 and find, wherein may contain 3-4 little effect mildew-resistance gene, and at hundred farmings 64 and capital couple 16 F of hybridizing
2:3Detect three QTL in the family, lay respectively on 2B, 2D and the 4D karyomit(e).
Summary of the invention
The method and the primer special thereof that the purpose of this invention is to provide a kind of powdery mildew resistant wheat auxiliary screening.
The primer special of powdery mildew resistant wheat auxiliary screening provided by the present invention is a primer among Xbarc79-1 and the Xwmc288-1 at least one pair of;
Described primer to the deoxyribonucleotide sequence of Xbarc79-1 shown in sequence in the sequence table 1 and sequence 2;
Described primer to the deoxyribonucleotide sequence of Xwmc288-1 shown in sequence in the sequence table 3 and sequence 4.
The present invention also provides a kind of method of powdery mildew resistant wheat auxiliary screening, comprise the steps: that the genomic dna with wheat to be measured is a template, at least one pair of primer with above-mentioned primer centering carries out pcr amplification, detect amplified production, the powdery-mildew-resistance wheat of the wheat to be measured of 235bp and/or 295bp band for the candidate arranged in the amplified production.
In the described pcr amplification, the PCR reaction system of per 15 μ l is as follows: the template DNA of 20ng, the dNTPs of 450 μ M, 0.95U Taq archaeal dna polymerase, 1.5 10 * PCR buffer of μ l, primer shown in 4pmol sequence 1, the sequence 2 is the totally 2.3 μ l of the primer shown in totally 2.3 μ l and/or 4pmol sequence 3, the sequence 4, with sterile distilled water postreaction system to 15 μ l.The program of described pcr amplification is: 94 ℃ of pre-sex change of elder generation 5 minutes; 94 ℃ of sex change are 1 minute then, 52 ℃ of annealing 1 minute, and 72 ℃ were extended totally 35 circulations 1 minute; Last 72 ℃ were extended 10 minutes.Described detection amplified production is amplified production to be carried out 6% denaturing polyacrylamide gel electrophoresis, and silver dyes colour developing then.
Application in the test kit of preparation powdery mildew resistant wheat auxiliary screening also belongs to protection scope of the present invention to described primer to Xbarc79-1 and/or Xwmc288-1.
The present invention also protects and contains the test kit of described primer to the powdery mildew resistant wheat auxiliary screening of Xbarc79-1 and/or Xwmc288-1.
Described primer all can be applicable in the wheat breeding Xbarc79-1 and/or Xwmc288-1, described method, described test kit.
The present invention also protects molecule marker Xbarc79 and/or the Xwmc288 of mildew-resistance gene QPm.caas-6BS; the deoxyribonucleotide sequence of described molecule marker Xbarc79 is shown in sequence in the sequence table 5, and the deoxyribonucleotide sequence of described molecule marker Xwmc288 is shown in sequence in the sequence table 6.Described mildew-resistance gene QPm.caas-6BS is positioned on the 6BS karyomit(e), and the nearest SSR in its both sides is labeled as Xbarc79 and Xwmc288, and genetic distance is 27.0cM between molecule marker Xbarc79 and the Xwmc288.Described molecule marker Xbarc79 and/or Xwmc288 can be detected under a plurality of envrionment conditionss, are stable molecule markers, and its effect is also particularly outstanding.Specifically, described molecule marker Xbarc79 can be obtained the increase genomic dna of hundred farmings 64 of Xbarc79-1 by described primer; Described molecule marker Xwmc288 can be obtained the increase genomic dna of hundred farmings 64 of Xwmc288-1 by described primer.Above-mentioned molecule marker can carry out assisting sifting to powdery mildew resistance gene in wheat.
Described molecule marker also can be applicable in powdery mildew resistant wheat auxiliary screening and/or the wheat breeding.
Described Powdery Mildew specifically can be the Powdery Mildew that the powdery mildew microspecies cause for E20 number.
Whether the present invention also protects a kind of auxiliary detection wheat to be measured for carrying the mildew-resistance gene method of wheat, comprise the steps: that with wheat cdna group DNA to be measured be template, with described primer Xbarc79-1 and/or Xwmc288-1 are carried out pcr amplification, detect the band whether 235bp and/or 295bp size are arranged in the amplified production; The wheat to be measured that the band of 235bp and/or 295bp size is arranged in the amplified production is candidate's the wheat that carries mildew-resistance gene; The band that had not both had the 235bp size in the amplified production, the wheat to be measured that does not also have the band of 295bp size is candidate's the wheat that does not carry mildew-resistance gene.
Primer provided by the present invention is to can be used for the clone of wheat anti-powdery mildew molecular breeding and mildew-resistance gene with molecule marker.Primer special of the present invention and molecule marker will play a significant role in wheat breeding for disease resistance.
Description of drawings
Figure 1A, Figure 1B are respectively the field histogram of maximum severity (MDS) of the two 16DH colonies in hundred farmings, 64 * capital Powdery Mildew and course of disease area under curve (AUDPC)
Fig. 2 A is the amplification of primer to Xbarc79-1 antagonism sense parent, anti-sense pond and strain system of DH colony
Fig. 2 B is the amplification of primer to Xwmc288-1 antagonism sense parent, anti-sense pond and strain system of DH colony
Fig. 3 is composite interval mapping method localized mildew-resistance gene graphic representation in the two 16DH colonies in hundred farmings, 64 * capital
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.
All primers are synthetic to be finished by Beijing AudioCodes biotech firm.The wheat lines that all are used in following examples is all preserved the center from country of Chinese Academy of Agricultural Sciences farm crop germplasm.
The molecule marker Xbarc79 of embodiment 1, mildew-resistance gene and the acquisition of Xwmc288 and primer special thereof
Wheat * corn hybridization method (Laurie and Bennett is passed through in hundred farmings 64 and capital two 16,1988, Theproduction of haploid wheat plants from wheat * maize crosses.Theor.Appl.Genet.76:393-397) obtains to contain the DH colony of 181 familys.
One, phenotypic acquisition and colony's field performance situation thereof
With in China's wheat at present popular Powdery Mildew microspecies E20 .1993 such as (, Establishment of wheatpowdery mildew isolates with putative virulence genotype.Plant Prot.19:27-28) Duan the two 16DH colonies in hundred farmings, 64 * capital of containing 181 familys carried out strain-forming period resistance respectively at 2005-06 and 2007-08 in Beijing and Anyang identify.The result shows that maximum severity (MDS) of the disease index of the two 16DH colonies in this hundred farmings, 64 * capital and course of disease area under curve (AUDPC) show as continuity and change in the field, is typical quantitative character heredity.The field histogram of maximum severity (MDS) of the two 16DH colonies in hundred farmings, 64 * capital Powdery Mildew and course of disease area under curve (AUDPC) as shown in Figure 1.Wherein, Figure 1A represents 2005-06 and 2 years three point (2005-06 Anyang points of 2007-08,2005-06 Beijing point and 2007-08 Beijing point) (what represent as first pillar among the figure is in whole DH colony to average MD S value, maximum severity (MDS) has 78 strain systems when 0-5%, each later pillar in like manner), Figure 1B represents to put two annual AUDPC values in Beijing, and (what represent as first pillar among the figure is in whole DH colony, course of disease area under curve (AUDPC) has 15 strain systems when 0-10%, each later pillar is in like manner) as can be seen from Figure 1, contain a plurality of mildew-resistance genes in hundred farmings 64.Select 5 extremely disease-resistant individual plants and 5 extremely susceptible individual plants for use according to the field investigation data, extract its DNA respectively, the DNA balanced mix of 5 extremely disease-resistant individual plants is formed disease-resistant pond, the DNA balanced mix of 5 extremely susceptible individual plants is formed susceptible pond, screening and the chain molecule marker of disease-resistant gene.
Two, the right acquisition of primer special
Select 406 pairs of SSR primers for use, comprising the 140 couples of BARC series primers .2002 such as (, Characterization of trinucleotide SSR motifs in wheat.Theor.Appl.Genet.104:286-293) Song, the 81 pairs of GWM series primers (
Deng .1998, A microsatellite map of wheat.Genetics 149:2007-2023), 134 couples of WMC series primer (.2003 such as Gupta, Transferable EST-SSRmarkers for the study of polymorphism and genetic diversity in bread wheat.Mol.Gen.Genet.270:315-323), 14 couples of GDM series primer (.2000 such as Pestsova, Isolation and mapping ofmicrosatellite markers specific for the D genome of bread wheat.Genome 43:689-697), 7 couples of CFA series primer (.2001 such as Sourdille, Improvement of the genetic maps of wheat usingnew microsatellite markers.Plant Anim.Geno.IX 167) and 30 couples of CFD series primer (Guyomarc ' .2002 such as h H, Studies of the transferability of microsatellites derived fromTriticum tauschii to hexaploid wheat and to diploid related species using amplification, hybridization and sequence comparisons.Theor.Appl.Genet.105:736-744), all primers are synthetic finishes by Beijing AudioCodes biotech firm.With above-mentioned primer to be used between the parent and anti-, sense pond between the polymorphism screening.
With hundred farmings 64, capital two 16 and the disease-resistant pond of above-mentioned steps one and the genomic dna in susceptible pond is template, carries out PCR and detects.The PCR reaction system of 15 μ l consists of: the template DNA of 20ng, the dNTPs of 450 μ M (available from sky root company), 0.95U Taq archaeal dna polymerase (available from sky root company), 1.5 10 * PCR buffer of μ l (available from sky root company), 4pmol upstream and downstream primer is totally 2.3 μ l, with sterile distilled water postreaction system to 15 μ l.The program of pcr amplification is: 94 ℃ of pre-sex change of elder generation 5 minutes; 94 ℃ of sex change are 1 minute then, 52 ℃ of annealing 1 minute, and 72 ℃ were extended totally 35 circulations 1 minute; Last 72 ℃ were extended 4 ℃ of preservations 10 minutes.
Pcr amplification product is carried out 6% denaturing polyacrylamide gel electrophoresis to be detected, step is as follows: get 15 μ l amplified productions, add 4 μ l sex change load sample indicator (98% no ion methane amides, 10mM EDTA pH8.0,0.1% tetrabromophenol sulfonphthalein and 0.1% dimethylbenzene green grass or young crops), behind 95 ℃ of sex change 5min, put into mixture of ice and water immediately and cool off 10min; The amplification sample 4.5 μ l of every part of sex change are electrophoretic separation in 6% the denaturing polyacrylamide gel in concentration, and silver dyes colour developing.
The result shows, above-mentioned primer centering has 7 pairs of primers between disease-resistant pond, susceptible pond and anti-, sense parent consistent difference being arranged.Detect disease-resistant strain and susceptible strain respectively with 7 pairs of primers selecting, and be positioned near the QTL peak value 2 pairs of primers showing and the disease-resistant gene close linkage.With these two pairs of primers to called after Xbarc79-1 and Xwmc288-1, primer to the deoxyribonucleotide sequence of Xbarc79-1 shown in sequence in the sequence table 1 and sequence 2, primer to the deoxyribonucleotide sequence of Xwmc288-1 shown in sequence in the sequence table 3 and sequence 4.
Three, the discovery of molecule marker Xbarc79 and Xwmc288 and evaluation
With disease-resistant parent (hundred farmings 64), susceptible parent (capital two 16), disease-resistant pond, susceptible pond and the strain of DH colony is as sample.Genomic dna with each sample is that template is carried out pcr amplification (the same step 2 of pcr amplification system and amplification program) with primer respectively to Xbarc79-1 and Xwmc288-1 respectively.Pcr amplification product is carried out the denaturing polyacrylamide electrophoresis, and silver dyes colour developing (the same step 2 of concrete operations).
Reclaim the band of 235bp and 295bp from denaturing polyacrylamide gel, carry out pcr amplification (the same step 2 of pcr amplification system and amplification program), amplified production obtains the band that size is 235bp and 295bp after reclaiming with 1.5% sepharose, the sequence called after Xbarc79 that will obtain Xbarc79-1 amplification with primer will be with the primer sequence called after Xwmc288 that amplification obtains to Xwmc288-1.Respectively Xbarc79 and Xwmc288 are checked order.Sequencing result shows that the deoxyribonucleotide sequence of Xbarc79 is shown in sequence in the sequence table 5, and the deoxyribonucleotide sequence of Xwmc288 is shown in sequence in the sequence table 6.
Primer resists the sense parent, resists the amplification of feeling pond and strain system of DH colony shown in Fig. 2 A Xbarc79-1, and primer resists the sense parent, resists the amplification of feeling pond and strain system of DH colony shown in Fig. 2 B Xwmc288-1.Wherein, M is a dna molecular amount standard, and Pr is hundred farmings 64 (disease-resistant parents), and Ps is two 16 (the susceptible parents) in capital, and Br is disease-resistant pond, and Bs is susceptible pond, and R is that disease-resistant strain is, S is that susceptible strain is.Black arrow is molecule marker Xbarc79 and the pairing fragment of Xwmc288 among the figure.
The result shows between parent and anti-sense pond polymorphism is arranged all at molecule marker Xbarc79 on the 6BS karyomit(e) and Xwmc288, shows that tentatively these molecule markers and mildew-resistance gene are chain.And hundred farmings 64, disease-resistant pond and disease-resistant strain system all can amplify the dna fragmentation of 235bp and/or 295bp; And all can not amplify the dna fragmentation of 235bp and/or 295bp in two 16, susceptible pond in Beijing and the susceptible strain system.
Four, the acquisition of linkage map
With molecule marker Xbarc79 and Xwmc288 181 strain systems of DH colony are carried out pcr amplification (the same step 2 of pcr amplification system and amplification program) respectively, with Mapmanager QTX b20 and Cartographer 2.5 softwares data are handled, these two molecule markers are carried out linkage analysis, make up linkage map.The result shows that these two molecule markers are all chain with mildew-resistance gene, and genetic distance is 27.0cM between the mark of its both sides, LOD value climax, and under different envrionment conditionss, the contribution rate that phenotypic variation is changed is 10.3-16.0%.Concrete result such as table 1 and shown in Figure 3.Among Fig. 3, curve 06A represents the MDS value of 2005-06 Anyang point, and curve 06B represents the MDS value of 2005-06 Beijing point, and curve 08B represents the MDS value of 2007-08 Beijing point, curve A verage represents the population mean of MDS, and curve A UDPC represents the mean value of 2 years AUDPC values of Beijing point.Be on the occasion of as can be seen from its additive effect, this mildew-resistance gene is from disease-resistant parent's hundred farmings 64.
Table 1. composite interval mapping method detects the mildew-resistance gene of the two 16DH colonies in hundred farmings, 64 * capital
The application of embodiment 2, molecule marker Xbarc79 and Xwmc288 and primer special thereof
One, whether homophyletic system and kind do not carry molecule marker Xbarc79 and/or Xwmc288 to detect wheat
The strain system of DH colony and the wheat breed genomic dna to be detected that are produced with two 16 filial generations of hundred farmings 64 and capital are template, adopt primer that Xbarc79-1 and Xwmc288-1 are carried out pcr amplification.
The PCR reaction system of 15 μ l consists of: the template DNA of 20ng, the dNTPs of 450 μ M (available from sky root company), 0.95U Taq archaeal dna polymerase (available from sky root company), 1.5 10 * PCR buffer of μ l (available from sky root company), the 4pmol primer is to Xbarc79-12.3 μ l, the 4pmol primer is to Xwmc288-12.3 μ l, with sterile distilled water postreaction system to 15 μ l.The program of pcr amplification is: 94 ℃ of pre-sex change of elder generation 5 minutes; 94 ℃ of sex change are 1 minute then, 52 ℃ of annealing 1 minute, and 72 ℃ were extended totally 35 circulations 1 minute; Last 72 ℃ were extended 4 ℃ of preservations 10 minutes.
Pcr amplification product is carried out 6% denaturing polyacrylamide gel electrophoresis to be detected, step is as follows: get 15 μ l amplified productions, add 4 μ l sex change load sample indicator (98% no ion methane amides, 10mM EDTA pH8.0,0.1% tetrabromophenol sulfonphthalein and 0.1% dimethylbenzene green grass or young crops), behind 95 ℃ of sex change 5min, put into mixture of ice and water immediately and cool off 10min; The amplification sample 4.5 μ l of every part of sex change are electrophoretic separation in 6% the denaturing polyacrylamide gel in concentration, and silver dyes colour developing.The result is as shown in table 3.
Two, detect wheat not homophyletic system and kind to the resistance of Powdery Mildew
The DH strain system and the wheat breed to be detected that are produced with two 16 filial generations of hundred farmings 64 and capital are experiment material, will carry out the strain-forming period resistance evaluation respectively at 2005-06 and 2007-08 in Beijing and Anyang for the examination wheat lines.100 wheat seeds of every row sowing adopt the completely random block design, and three repetitions are established in test, the single file district, and long 1.5 meters of row is every the susceptible contrast of ten behavior delegation (capital two 16).Become the strain phase at wheat, when two 16 morbidities in susceptible contrast wheat capital are abundant, the investigation incidence.Each for the examination material to the disease resistance of Powdery Mildew with maximum severity (MDS) expression, maximum severity (MDS) adopts 4 grade standards (seeing Table 2), promptly high anti-, in anti-, middle sense and high sense.Detected result sees Table 3.
Table 2 wheat powdery mildew becomes maximum severity (MDS) grade scale of strain phase
| Resistance | MDS | Symptom |
| High anti- | 0-5％ | White powder sorus area accounts for down the percentage of the two leaf total areas less than 5% |
| In anti- | 5-20％ | White powder sorus area accounts for down the percentage of the two leaf total areas between 5-20%; |
| Middle sense | 20-40％ | White powder sorus area accounts for down the percentage of the two leaf total areas between 20-40% |
| High sense | 40-100％ | White powder sorus area accounts for down the percentage of the two leaf total areas greater than 40% |
The detected result that table 3 with molecule marker Xbarc79 and Xwmc288 to different wheat strains is
Annotate :+expression contains mildew-resistance gene, and-expression does not contain mildew-resistance gene.Wheat with the L beginning in the table 3 is the DH strain system that two 16 filial generations of hundred farmings 64 and capital are produced, all the other wheat lines are once or at present to produce the known disease-resistant or susceptible wheat breed of promoting, and all can obtain from country of Chinese Academy of Agricultural Sciences farm crop germplasm preservation center.
Experimental result shows: the wheat strain that contains the mildew-resistance ospc gene ties up to the field and all shows as mildew-resistance, and all can amplify the band of 235bp and/or 295bp.
Sequence table
＜110〉Institute of Crop Science, Chinese Academy of Agricultural Science
＜120〉a kind of method of powdery mildew resistant wheat auxiliary screening and primer special thereof
<130>CGGNARZ92264
<160>6
<210>1
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
gcgttggaaa?ggaggtaatg?ttagatag 28
<210>2
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
tcgtgggtta?caagtttggg?aggtca 26
<210>3
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
aaccaaaacc?ctaatgtcac?ct 22
<210>4
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
tagagcatgt?gtggtgtgag?at 22
<210>5
<211>235
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
gcgttggaaa?ggaggtaatg?ttagatagat?agatagatag?atagatagat?agatagatag 60
atagatagat?agatagatag?atagatagat?agatagagat?atatatatat?atatgtgtgt 120
gtgtgtgttg?tattcaaact?tctatctcct?gtctctctct?ctctctctct?ctctctctct 180
ctctctcgtg?taactctgga?gagtcctagt?gacctcccaa?acttgtaacc?cacga 235
<210>6
<211>295
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>6
aaccaaaacc?ctaatgtcac?ctagatactg?taatgtcatc?gcgggtatga?ttatacgata 60
tgcatcacac?agtctcagat?tcatctattc?aaacgaacac?aaagaacttc?aaaggccccc?120
cgcccacaca?aacacacaca?cacactaaga?ctccatcaca?cacaccatct?ctctctctct?180
ctctctcaca?cacacacaca?cacacacaca?cacccacacc?cacacccaca?cccacaaata?240
cacacacaca?cacacacaca?cactaagtac?tccatctcac?accacacatg?ctcta 295
Claims (7)
1. the method for a powdery mildew resistant wheat auxiliary screening, comprise the steps: that the genomic dna with wheat to be measured is a template, with primer Xbarc79-1 is carried out pcr amplification, detect amplified production, the powdery-mildew-resistance wheat of the wheat to be measured of 235bp band for the candidate arranged in the amplified production;
Described primer to the deoxyribonucleotide sequence of Xbarc79-1 shown in sequence in the sequence table 1 and sequence 2.
2. method according to claim 1, it is characterized in that: in the described pcr amplification, the PCR reaction system of per 15 μ l is as follows: the template DNA of 20ng, the dNTPs of 450 μ M, 0.95U Taq archaeal dna polymerase, 1.5 10 * PCR buffer of μ l, the primer shown in 4pmol sequence 1, the sequence 2 be the totally 2.3 μ l of the primer shown in totally 2.3 μ l and 4pmol sequence 3, the sequence 4, with sterile distilled water postreaction system to 15 μ l:
The program of described pcr amplification is: 94 ℃ of pre-sex change of elder generation 5 minutes; 94 ℃ of sex change are 1 minute then, 52 ℃ of annealing 1 minute, and 72 ℃ were extended totally 35 circulations 1 minute; Last 72 ℃ were extended 10 minutes;
Described detection amplified production is amplified production to be carried out 6% denaturing polyacrylamide gel electrophoresis, and silver dyes colour developing then.
3. primer is to the application of Xbarc79-1 in the test kit of preparation powdery mildew resistant wheat auxiliary screening;
Described primer to the deoxyribonucleotide sequence of Xbarc79-1 shown in sequence in the sequence table 1 and sequence 2.
4. contain the test kit of primer to the powdery mildew resistant wheat auxiliary screening of Xbarc79-1;
Described primer to the deoxyribonucleotide sequence of Xbarc79-1 shown in sequence in the sequence table 1 and sequence 2.
5. claim 1 or 2 described methods, the application of the described test kit of claim 4 in powdery mildew resistant wheat auxiliary screening.
6. the molecule marker Xbarc79 of mildew-resistance gene, its deoxyribonucleotide is shown in sequence in the sequence table 5.
7. the application of the described molecule marker of claim 6 in powdery mildew resistant wheat auxiliary screening.
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| CN102533750B (en) * | 2012-02-20 | 2013-03-13 | 中国科学院成都生物研究所 | Wheat-powdery-mildew-resistance-associated gene molecular marker primers and use thereof |
| CN111996283B (en) * | 2020-09-22 | 2022-09-27 | 烟台大学 | Molecular marker closely linked with wheat powdery mildew resistance gene PmLS5082 and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1428440A (en) * | 2002-09-27 | 2003-07-09 | 天津师范大学 | Molecular marker linked with wheat mildew-resistance gene |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1428440A (en) * | 2002-09-27 | 2003-07-09 | 天津师范大学 | Molecular marker linked with wheat mildew-resistance gene |
Non-Patent Citations (3)
| Title |
|---|
| Daryl Somers.SSRs from the wheat microsatellite consortium..《GrainGenes》.2004, * |
| Song QJ et al..Development and mapping of microsatellite(SSR) markers in wheat(supp3.《Theor Appl Genet》.2005, * |
| 王竹林.小麦慢性病的遗传育种研究进展.《麦类作物学报》.2006,第26卷(第1期), * |
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