CN108300799A - The high-throughput detection label of wheat powdery mildew resistant gene Pm 5 e and its application in breeding - Google Patents
The high-throughput detection label of wheat powdery mildew resistant gene Pm 5 e and its application in breeding Download PDFInfo
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Abstract
The invention discloses two single nucleotide polymorphism molecular labelings that can be used for high-throughput marker assisted selection breeding of powdery mildew resistant gene Pm 5 e and its application processes in breeding.To China, powdery mildew microspecies show as highly resistance or are immunized Pm5e currently popular, have important utility value in breeding.The two detection labels are KASP labels, quick, efficient, high-throughput it can detect distribution of the wheat powdery mildew resistant gene Pm 5 e in wheat breed and the distribution situation in segregating population, be conducive to Pm5e finely positionings and clone, and assisted Selection can be marked to Pm5e in breeding population, the powder mildew resistance of wheat is improved using Pm5e, the influence to wheat yield is reduced, wheat anti-powdery mildew breeding efficiency is improved.
Description
Technical field
The present invention relates to crop molecular mark technical fields, and in particular to a kind of powdery mildew resistance gene in wheat
The high-throughput detection label of Pm5e and its application in breeding.
Background technology
Wheat (Triticum aestivum L.) is the second largest cereal crops that China is only second to rice, long-term growing surface
Product accounts for about 27% or so of the cereal crops gross area at 2666.67 ten thousand mu or more.Thereby it is ensured that yield and quality of wheat is steady
It is to be related to the key factor that China's grain security and people's living standard are continuously improved that step, which improves,.
Wheat powdery mildew is a kind of worldwide disease, is globally distributed the larger area of various regions, especially humidity and occurs more
It is serious.General underproduction 5%-10% when morbidity, 20% or more the grave illness field underproduction.In recent years, the morbidity range of wheat powdery mildew
Constantly expanding, the extent of injury also constantly aggravates, it has also become current Wheat Production seriously threatens.Selection and breeding and use are efficient anti-
Sick kind is prevention powdery mildew economy, safe and effective measure.And the basis of powdery mildew disease-resistant breeding is efficient, diversified
Anti- source.The Resistant expression and genetic characteristics for furtheing investigate disease-resistant gene will be helpful to efficiently use these disease-resistant genes.
Different wheat breed resistances is different, and contained disease-resistant gene is also different.Up to now, located and give definite designation
Quality mildew-resistance gene has 86, i.e. Pm1-Pm60, wherein Pm1, Pm2, Pm3, Pm4, Pm5 and Pm24 have 5 respectively, 2,17,
4,6 and 2 allele, Pm18=Pm1c, Pm22=Pm1e, Pm23=Pm4c, Pm31=Pm21.This 86 allele positions
In on 56 chromosomal focis of 21 chromosome.Wherein, Pm5 genes are located on 7BL chromosomes, contain 6 allele, respectively
For Pm5a, Pm5b, Pm5c, Pm5d, Pm5e and Pm5f.
Pm5e is a recessive mildew-resistance gene, carries the winter wheat variety Fu Zhuang 30 of the gene, is passed through from Jingyang 30
Made of systematic breeding, to China, powdery mildew microspecies show as highly resistance or are immunized currently popular, have important utility value.
Forefathers, to the assignment of genes gene mapping, are tentatively located on 7BL chromosomes using SSR marker, but the two SSR markers and target
The genetic distance of gene is respectively 11cM and 6.6cM (Huang etc. 2003), and just equal (2008) on this basis, develop 2 to Wang Hong
A SSR marker, and genetic distance is respectively 5.1cM and 4.9cM, although the two SSR markers shorten to a certain extent
Genetic distance between Pm5e genes and label, but there are still genetic distances farther out, polymorphism level is relatively low, detection efficiency
The problems such as low；Later Liu Zhi bravely waits (2008) to filter out specific SNP site by BSR-Seq, and is converted to drawing for specificity
Object, further to the Pm5e assignments of genes gene mapping, by the Pm5e assignments of genes gene mapping between EST marks CJ729392 and CJ584170, the research
Primer specificity is improved, but polymorphism level is still relatively low, and the genetic distance between 2 labels is larger (23.5cM).
Competitive polymorphic allele PCR (Kompetitive Allele Specific PCR, KASP) is current state
One of the main stream approach of Genotyping analysis is carried out on border to single SNP, principle is the special matching according to prime end base
Parting is carried out to SNP and InDel, including 2 terminal bases are the forward primer and 1 reverse primer of different (i.e. two kinds of SNP),
Two ends of forward primer 5 ' carry not homotactic fluorescent linker sequence FAM or HEX respectively, therefore, according to fluorescence signal
The specificity of different just detectable SNP.KASP labels can carry out SNP site accurately diallele and judge, Ke Yitong
When great amount of samples is detected, while also having many advantages, such as that genetic stability is good, be a kind of molecular labeling of high throughput.
Therefore, filter out with the relevant SNP of wheat powdery mildew resistance, and be further developed as being suitable for it is high-throughput,
The KASP of high efficient detection is marked, and is applied to the selection of resist powdery mildew of wheat material, to improving yield and quality of wheat etc.
There is important meaning.
Invention content
For the above-mentioned prior art, the object of the present invention is to provide a kind of high throughput inspections of wheat powdery mildew resistant gene Pm 5 e
Mark remembers and its application in breeding.
To achieve the above object, the present invention adopts the following technical scheme that：
The first aspect of the present invention provides a kind of and relevant SNP marker of wheat powdery mildew resistance, includes that 2 SNP are marked altogether
Note respectively marks AX-95000860 and label AX-94638908；The label AX-95000860 and AX-94638908 is equal
On wheat 7BL chromosomes, wherein it is 721220493 to mark the physical location of AX-95000860, and base is at SNP site
C or T；It is 708115367 to mark the physical location of AX-94638908, and base is C or T at SNP site.
Application of the above-mentioned SNP marker in wheat anti-powdery mildew breeding is also protection scope of the present invention.
The second aspect of the present invention, provide it is a kind of based on KASP technological development for detect and powdery mildew resistant gene Pm 5 e
The primer of the SNP of close linkage；Described and powdery mildew resistant gene Pm 5 e close linkage SNP is respectively AX-95000860 and AX-
94638908；The physical location of AX-95000860 is 721220493, and base is C or T at SNP site；Molecular labeling AX-
94638908 physical location is 708115367, and base is C or T at SNP site；
Wherein, the primer sequence of AX-95000860 is detected respectively as shown in SEQ ID NO.1-3；Detect AX-94638908
Primer sequence respectively as shown in SEQ ID NO.4-6.
The third aspect of the present invention provides detection reagent or kit containing above-mentioned primer.
The present invention also provides the application of above-mentioned primer or detection reagent or kit in the initiative of wheat anti-powdery mildew material.
The present invention also provides above-mentioned primers or detection reagent or kit in molecular labeling auxiliary wheat anti-powdery mildew selection
Application in breeding.
The present invention also provides above-mentioned primers or detection reagent or kit to have the wheat resource of powder mildew resistance in selection and breeding
In application.
The fourth aspect of the present invention provides a kind of method of detection and the SNP of powdery mildew resistant gene Pm 5 e close linkage, packet
Include following steps：
(1) DNA of wheat samples to be measured is extracted；
(2) mix primer or SEQ ID shown in 3 μ l, the SEQ ID NO.1-3 of template DNA of a concentration of 20ng/ μ l are taken
0.0825 μ l, 2 × Master Mix of mix primer, 3 μ l shown in NO.4-6 carry out PCR amplification；
(3) fluorescence detector is used to analyze pcr amplification product genotype.
Preferably, in step (2), the condition of PCR amplification is：
1) 94 DEG C of pre-degeneration 5min；
2) 94 DEG C of denaturation 20s；
3) 65 DEG C of annealing 30s, step 2) -3) it recycles 10 times, each cycle annealing temperature reduces by 0.8 DEG C；
4) 94 DEG C of denaturation 20s；
5) 57 DEG C of annealing 30s, step 4) -5) it recycles 38 times；
6) 4 DEG C of preservations.
Beneficial effects of the present invention：
The present invention provides two have in breeding important utility value wheat powdery mildew resistant gene Pm 5 e SNP marker
(label AX-95000860 and label AX-94638908), and develop KASP detections based on the two newfound SNP markers
Label quick, efficient, high-throughput can detect distributions of the Pm5e in wheat breed and the distribution situation in segregating population,
Be conducive to Pm5e finely positionings and clone, and assisted Selection can be marked to Pm5e in breeding population, is carried using Pm5e
The powder mildew resistance of high wheat reduces the influence to wheat yield, improves wheat anti-powdery mildew breeding effect
Description of the drawings
Fig. 1：The KASP genotyping results of AX-95000860 labels represent C (mildew-resistance) genotype close to horizontal axis dot,
Longitudinal axis dot represents T (sense powdery mildew) genotype, and cornerwise dot represents heterozygous；The holes DNA are not added for × representative, and ■ is represented
ddH2O blank controls.
Fig. 2：The KASP genotyping results of AX-94638908 labels represent C (mildew-resistance) genotype close to horizontal axis dot,
Longitudinal axis dot represents T (sense powdery mildew) genotype, and the dot near diagonal line represents heterozygous；The holes DNA, ■ are not added for × representative
Represent ddH2O blank controls.
Specific implementation mode
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
As described in background technology, filter out with the relevant SNP of wheat powdery mildew resistance, and developed be energy
The KASP detection labels of its accurate SNP site, the selection to resist powdery mildew of wheat material improve yield and quality of wheat etc.
There is important meaning.KASP to develop Pm5e detects label, and the present invention utilizes 35K biochip technologies, anti-by building
Sick pond and susceptible pond filter out the probe there are polymorphic SNP site between disease-resistant pond and susceptible pond, for searching for wheat cdna
Data unit sequence library finds out comparison to the probe on 7BL chromosomal focis, amounts to 88.The research knot announced according to forefathers
Fruit, Pm5e genes are positioned between two est sequences CJ729392 and CJ584170, the heredity between the two est sequences
Distance is 23.5cM.Using comparison result, the specific probe between the two est sequences is selected, positioned at the two
SNP is converted to KASP labels by the probe containing SNP specific positions between est sequence through further comparing analysis,
Fu Zhuang 30 containing powdery mildew resistant gene Pm 5 e hybridizes 214 F obtained with high sense powdery mildew material C hancellor2:3Dai Qun
Genotyping is carried out in body, by linkage analysis, is found two KASP labels closer with Pm5e gene linkages, be can be used for Pm5e
High-throughput detection and marker-assisted breeding.
The molecule mark of the two provided by the invention wheat powdery mildew resistant gene Pm 5 es for having important utility value in breeding
Note, respectively：Molecular labeling AX-95000860 and molecular labeling AX-94638908；The two equal positions molecular labeling (i.e. SNP)
In on wheat 7BL chromosomes, wherein the physical location of molecular labeling AX-95000860SNP is 721220493, at SNP site
Base is C or T；The physical location of molecular labeling AX-94638908SNP is 708115367, and base is C or T at SNP site.
(positions SNP are the determinations according to the wheat cdna sequence library announced).
SNP marker AX-95000860 and label AX-94638908 systems are present invention firstly discovers that and propose, the two SNP marks
The two SNP markers, are applied to the transformation of Pm5e genes by note and Pm5e gene close linkages, for accelerating powdery-mildew-resistance wheat
The selection and breeding of kind, improving breeding efficiency has important practice significance.
To detect the two specificity of key SNP in Wheat in China powdery mildew group, be conducive to mildew-resistance base
Because of Pm5e finely positionings and clone, the present invention is based on KASP technological development closely to connect for detecting with powdery mildew resistant gene Pm 5 e
The primer of the SNP of lock, primer sequence are as follows：
AX-95000860-KASP-FAM_C-R：5 ＇ CAGGATTGGACTCGGCTGGAAAC3 ＇, SEQ ID NO.1；
AX-95000860-KASP-HEX_T-S:5 ＇ CAGGATTGGACTCGGCTGGAAAT3 ＇, SEQ ID NO.2；
AX-95000860-KASP-R:5 ＇ ATGTCAGGTCACCACGATGC3 ＇, SEQ ID NO.3.
AX-94638908-KASP-FAM_C-R：5 ＇ ATGATAACATGCTGCGCATGAC3 ＇, SEQ ID NO.4；
AX-94638908-KASP-HEX_T-S:5 ＇ ATGATAACATGCTGCGCATGAT3 ＇, SEQ ID NO.5；
AX-94638908-KASP-R:5 ＇ TACACAAACTAGGTGGAGGTACAAC3 ＇, SEQ ID NO.6.
After obtaining above-mentioned primer, it is as follows that inventor further provides specific detection method：
1. extracting wheat complete genome DNA to be detected using CTAB methods；
2. pair Fu Zhuang 30 hybridizes 214 F obtained with Chancellor2Group carries out powdery mildew phenotypic evaluation, chooses high
The F of anti-, high sense2Each 15 plants of single plant is blended together anti-pond and sense pond, the SNP near target gene is found using 35K genetic chips respectively
Site；
3. utilizing primer5.0, these SNP sites are converted to the genomic DNA of KASP detections label and the extraction of upper step
PCR amplification is carried out on Q-Cycler 96PCR instrument (Hain Life science UK ltd.UK), and in ABI Quant
StudioTM(Life Technologies Corporation USA) is carried out in 12K Flex real-time fluorescence quantitative PCR systems
SNP is detected；
The standard of judgement is determined with the color put with the position for being gathered in horizontal axis or the longitudinal axis：Signal is red, is gathered in cross
It compares near axis and with mildew-resistance and flocks together, be determined as mildew-resistance gene type；Signal is blue, white with the longitudinal axis and sense
The control of powder disease flocks together, and is judged to feeling powdery mildew gene type；Signal is green, is compareed with two reference axis and resistant, susceptible
It does not flock together, the judgement near y=x straight lines is heterozygous genotypes；
4. analysis is above-mentioned to obtain the distribution situation with the SNP marker of Pm5e gene close linkages in wheat population.
The specific method is as follows：
Wheat complete genome DNA to be detected is extracted using CTAB methods：
(a) it takes wheat young leaflet tablet to be placed in 96 hole deep-well plates, is handled using liquid nitrogen frozen and in being ground in tissue grinder
At powder；(b) each Kong Zhongjia in 96 hole deep-well plates preheats 65 DEG C of 800 μ L of CTAB buffer solutions, is placed in 65 DEG C of water-baths
90min gently shakes once every 10min, DNA is made fully to crack；(c) it is 24 that 800 μ L volume ratios, which are added,:1 chloroform isoamyl
Alcohol mixed liquor, gently shakes 10min；(d) 12000r centrifuges 10min under conditions of 4 DEG C, then takes 600 μ L supernatants, be placed in new
(notice that serial number corresponds) in clean 96 hole deep-well plates；(e) add the isopropanol and 60 μ L 3M sodium acetates (pH=of 600 μ L precoolings
5.2) light shake shakes up, you can sees that white DNA floccules generate, is placed in -20 DEG C of refrigerator freezing 60min, increases DNA output.(f)
Supernatant is outwelled after 4000r centrifugations 10min, is air-dried to alcohol-free taste after 70% ethyl alcohol of precipitation precooling is cleaned 2-3 times；
(g) plus 200 μ L ultra-pure water dissolving DNAs are placed in -20 DEG C of refrigerators and save backup after DNA is substantially soluble in water.
Marker development is detected with the KASP of Pm5e gene linkages：
Currently, have issued for the label of 6 and Pm5e close linkages, including 4 SSR markers (Xgwm783,
Xgwm1267, Xwmc364 and Xbarc065) and 2 EST labels (CJ729392 and CJ584170).In disease-resistant parent's Fu Zhuang 30
Containing Pm5e genes, and another parent Chancellor shows as high sense powdery mildew.F is created using the two anti-sense materials2Point
Peel off body, totally 214 plants, and in seedling stage and Adult plant carry out powdery mildew phenotypic evaluation.According to qualification result, choose respectively highly resistance,
The F of height sense2Each 15 plants of single plant, structure is anti-, feels gene pool.The polymorphism SNP of sense gene pool is fought using 35K biochip technologies
Site is identified, wherein identifying 88 SNP sites on 7BL.Using the websites URGI in the reference gene group of China spring
Probe sequence comprising SNP site and two EST flags sequence are compared, 27 is filtered out and is located at the two EST labels
Between probe, be designed to KASP primers with primer5.0.In 214 F2Genotyping is carried out in segregating population, finds two
With the SNP marker of Pm5e gene linkages, and be further developed as KASP detection label.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
Test material used in the embodiment of the present invention is the test material of this field routine, can pass through commercial channel
It is commercially available.What is do not elaborated in the method applied in the present invention is state of the art.
Embodiment 1：Wheat volatiles DNA extractions
1) DNA extracts the collection of blade
In wheat tri-leaf period, wheat young leaflet tablet is chosen, is put by number in the deep-well plates of corresponding 96 hole, to prevent DNA
Degradation, operates on ice chest；
2) CTAB methods extraction Wheat volatiles DNA is utilized；- 20 DEG C are placed in save backup.
Above-mentioned steps are specific as follows：
(a) it takes wheat young leaflet tablet to be placed in 96 hole deep-well plates, is handled using liquid nitrogen frozen and in being ground in tissue grinder
At powder；(b) each Kong Zhongjia in 96 hole deep-well plates preheats 65 DEG C of 600 μ L of CTAB extracting solutions, is placed in 65 DEG C of water-baths
60min gently shakes once every 10min, DNA is made fully to crack；(c) it is 24 that 600 μ L volume ratios, which are added,:1 chloroform isoamyl
Alcohol mixed liquor, gently shakes 10min；(d) 12000r centrifuges 10min under conditions of 4 DEG C, then takes 500 μ L supernatants, be placed in new
(notice that serial number corresponds) in clean 96 hole deep-well plates；(e) add the isopropanol and 50 μ L 3M sodium acetates (pH=of 500 μ L precoolings
5.2) light shake shakes up, you can sees that white DNA floccules generate, is placed in -20 DEG C of refrigerator freezing 20min, increases DNA output.(f)
Supernatant is outwelled after 4000r centrifugations 10min, is air-dried to alcohol-free taste after 70% ethyl alcohol of precipitation precooling is cleaned 2-3 times；
(g) plus 200 μ L ultra-pure water dissolving DNAs are placed in -20 DEG C of refrigerators and save backup after DNA is substantially soluble in water.
The preparation method of used solution is as follows in above-mentioned steps：
(a) preparation of CTAB extracting solutions
Deionized water is added to be settled to 1000ml.
(b) 3M NaAc (pH=5.2)
Claim 40.8g sodium acetate trihydrates, pours into beaker, add water to 80ml, with glacial acetic acid tune pH value to 5.2, constant volume arrives
100ml。
(c) 1M Tris-HCl (pH=8.0)
121.1g Tris (or 157.6g Tris HCl) are dissolved in 800ml water, are cooled to room temperature, enriching hydrochloric acid (or
It is NaOH) adjust pH value to 8.0, (about needing 4.2ml) is settled to 1000ml.
(d) 0.5M EDTA2Na (pH=8.0)
Add 186.1g EDTA2Na in 800ml water, pH meter is put into solution, (20g is about needed with NaOH tune pH
NaOH particles), it is stirred while adding NaOH, EDTA2Na is made to dissolve, pH is about adjusted to 7.8 solution and becomes clarification, is settled to
1000ml。
(e) chloroform/isoamyl alcohol (24：1)
According to volume ratio chloroform：Isoamyl alcohol=24:1 prepares.
(f) 70% alcohol 1000ml
Add the deionized water of 300ml in the absolute ethyl alcohol of 700ml.
Embodiment 2：The exploitation that close linkage marks in Pm5e genes in Fu Zhuang 30
Currently, have issued for the label of 6 and Pm5e close linkages, including 4 SSR markers (Xgwm783,
Xgwm1267, Xwmc364 and Xbarc065) and 2 EST labels (CJ729392 and CJ584170).It is special due to SSR marker
Property it is relatively low, therefore the KASP detection labels of this research and development are completed on the basis of two EST are marked.Disease-resistant material Fu Zhuang 30
In contain Pm5e genes, Chancellor shows as high sense powdery mildew.F is created using the two anti-sense parents2Segregating population, altogether
214 plants, and in seedling stage and Adult plant carry out powdery mildew phenotypic evaluation.According to qualification result, highly resistance, the high F felt are chosen respectively2It is single
Each 15 plants of strain, structure is anti-, feels gene pool.It is identified using the SNP site of 35K biochip technologies confrontation sense gene pool,
In 88 SNP sites are identified on 7BL.Using the websites URGI to including SNP site in the reference gene group of China spring
Probe sequence and two EST flags sequence are compared, and filter out 27 probes being located between the two EST labels, use
Primer5.0 is designed to KASP detection labels.Rejected when design have hairpin structure, primer dimer can be formed between primer, on
Tm values are more than 3 DEG C of label between downstream primer, 16 are designed altogether, by F2Genotyping is carried out in segregating population, there are 3
KASP detection labels can be good at distinguishing the genotype of each individual, be found by linkage analysis, only the two of this patent announcement
A KASP detections label and Pm5e gene close linkages.
Embodiment 3：Primer is diluted to be mixed with KASP chemical examination primers：
After three primers of AX-95000860 are diluted to 100 μM with Tris HCl, according to volume ratio AX-95000860-
KASP-FAM_C-R：AX-95000860-KASP-HEX_T-S：AX-95000860-KASP-R：Tris HCl=6：6：15：23
Ratio mixing, after packing in -20 DEG C preservation, as KASP chemically examine primer.
The dilution of AX-94638908 primers and the same AX-95000860 of mixed method.
Embodiment 4：PCR amplification system and program
PCR system is prepared
System, 3 μ L (20ng/ μ L or so) template DNA, 2 × Master mix, 3 μ L (LGC are prepared on ice according to following table
Group UK), KASP chemically examines 0.0825 μ L of primer (primer is provided by matching your scientific and technological (China) Co., Ltd of silent winged generation).
PCR programs are as follows：
1) 94 DEG C of pre-degeneration 5min；
2) 94 DEG C of denaturation 20s；
3) 65 DEG C of annealing 30s (each cycle reduces by 0.8 DEG C), step 2) -3) it recycles 10 times；
4) 94 DEG C of denaturation 20s；
5) 57 DEG C of annealing 30s, step 4) -5) it recycles 38 times；
6) 4 DEG C of preservations.
Embodiment 5：Interpretation of result
Utilize ABI Quant StudioTM12K Flex real-time fluorescence quantitative PCRs systems to the PCR system that has expanded into
The collection of row SNP distribution results, the distribution results of AX-95000860 are as shown in Figure 1, wherein and horizontal axis is mildew-resistance gene type,
The longitudinal axis is sense powdery mildew gene type, hybridizes F with chancellor to Fu Zhuang 302For group 214 materials be detected after, obtain
To mildew-resistance gene proximate matter material 48 (identical with mildew-resistance of the present invention control Fu Zhuang 30 genotype), powdery mildew gene type is felt
Material 51 (identical with sense powdery mildew of the present invention control Chancellor genotype), heterozygous genotypes material 112 (and this hair
Bright heterozygous genotypes anti-, sense powdery mildew control parent is different), genotype is not detected because of DNA cause for quality in 3 materials.
The distribution results of AX-94638908 are as shown in Fig. 2, equally hybridize Fu Zhuang 30 with chancellor the 214 of F2 generations
A crowd surveillance analysis obtains 56, mildew-resistance gene proximate matter material, 47, sense powdery mildew gene proximate matter material, heterozygous genes proximate matter
111, material, genotype is not detected because of DNA cause for quality in 2 materials.According to the result combination powdery mildew phenotypic evaluation as a result,
It is genotype by Phenotypic Change, carries out the drafting of linkage map, it is for further study, help speed up the finely positioning of the gene
With clone.
The foregoing is merely the preferred embodiments of the application, are not intended to limit this application, for the skill of this field
For art personnel, the application can have various modifications and variations.Within the spirit and principles of this application, any made by repair
Change, equivalent replacement, improvement etc., should be included within the protection domain of the application.
SEQUENCE LISTING
<110>Shandong Agricultural University
<120>The high-throughput detection label of wheat powdery mildew resistant gene Pm 5 e and its application in breeding
<130> 2018
<160> 6
<170> PatentIn version 3.5
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<212> DNA
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caggattgga ctcggctgga aac 23
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caggattgga ctcggctgga aat 23
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<212> DNA
<213>Artificial sequence
<400> 3
atgtcaggtc accacgatgc 20
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<213>Artificial sequence
<400> 4
atgataacat gctgcgcatg ac 22
<210> 5
<211> 22
<212> DNA
<213>Artificial sequence
<400> 5
atgataacat gctgcgcatg at 22
<210> 6
<211> 25
<212> DNA
<213>Artificial sequence
<400> 6
tacacaaact aggtggaggt acaac 25
Claims (9)
1. a kind of and relevant SNP marker of wheat powdery mildew resistance, which is characterized in that include altogether 2 SNP markers, respectively mark
Remember AX-95000860 and label AX-94638908；Base at the label AX-95000860 and AX-94638908 SNP sites
It is C or T.
2. application of the molecular labeling described in claim 1 in wheat anti-powdery mildew breeding.
3. it is a kind of based on KASP technological development for detect and the primer of the SNP of powdery mildew resistant gene Pm 5 e close linkage,
It is characterized in that, described and powdery mildew resistant gene Pm 5 e close linkage SNP is respectively AX-95000860 and AX-94638908；
Wherein, the primer sequence of AX-95000860 is detected respectively as shown in SEQ ID NO.1-3；Detection AX-94638908's draws
Object sequence is respectively as shown in SEQ ID NO.4-6.
4. the detection reagent containing primer described in claim 3 or kit.
5. the detection reagent described in primer or claim 4 or kit described in claim 3 are in wheat anti-powdery mildew material
Application in initiative.
6. the detection reagent described in primer or claim 4 or kit described in claim 3 assist wheat in molecular labeling
Application in mildew-resistance selection and use.
7. the detection reagent described in primer or claim 4 or kit described in claim 3 are anti-with powdery mildew in selection and breeding
Application in the wheat resource of property.
8. a kind of detection method of detection and the SNP of powdery mildew resistant gene Pm 5 e close linkage, which is characterized in that including walking as follows
Suddenly：
(1) DNA of wheat samples to be measured is extracted；
(2) mix primer or SEQ ID NO.4- shown in 3 μ l, the SEQ ID NO.1-3 of template DNA of a concentration of 20ng/ μ l are taken
0.0825 μ l, 2 × Master Mix of mix primer, 3 μ l shown in 6 carry out PCR amplification；
(3) fluorescence signal for using fluorescence detector detection pcr amplification product, carries out Genotyping.
9. according to the method described in claim 8, it is characterized in that, in step (2), the condition of PCR amplification is：
1) 94 DEG C of pre-degeneration 5min；
2) 94 DEG C of denaturation 20s；
3) 65 DEG C of annealing 30s, step 2) -3) it recycles 10 times, each cycle annealing temperature reduces by 0.8 DEG C；
4) 94 DEG C of denaturation 20s；
5) 57 DEG C of annealing 30s, step 4) -5) it recycles 38 times；
6) 4 DEG C of preservations.
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| CN109295249A (en) * | 2018-10-28 | 2019-02-01 | 山东农业大学 | One wheat powdery mildew strain-forming period resistance QTL and its molecular labeling |
| CN109295249B (en) * | 2018-10-28 | 2021-08-03 | 山东农业大学 | Wheat powdery mildew adult-plant-stage resistance QTL and molecular marker thereof |
| CN109468409A (en) * | 2019-01-04 | 2019-03-15 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | A kind of powdery mildew resistance gene in wheat Pm2b high throughput detection label and its application |
| CN109468409B (en) * | 2019-01-04 | 2022-04-15 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | High-throughput detection marker for wheat powdery mildew resistance gene Pm2b and application thereof |
| CN111763249A (en) * | 2019-03-08 | 2020-10-13 | 中国科学院遗传与发育生物学研究所 | Plant powdery mildew resistance related protein Pm5e, and coding gene and application thereof |
| CN111719008A (en) * | 2019-03-19 | 2020-09-29 | 中国科学院遗传与发育生物学研究所 | SNP coseparated with wheat powdery mildew disease-resistant gene Pm5e and application thereof |
| CN113234852A (en) * | 2021-06-30 | 2021-08-10 | 四川省农业科学院作物研究所 | Molecular marker and primer group for identifying wheat powdery mildew resistance and application |
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