CN105087760B - The labeled primer of powdery mildew resistance gene in wheat Stpk-V a kind of and application - Google Patents
The labeled primer of powdery mildew resistance gene in wheat Stpk-V a kind of and application Download PDFInfo
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Abstract
The present invention provides a kind of powdery mildew resistance gene in wheatStpk‑VLabeled primer and its in resist powdery mildew of wheatStpk‑VApplication in genetic test, labeled primer InDelw190By nucleotide sequence such as SEQ ID NO:Sense primer and such as SEQ ID NO shown in 1：Anti-sense primer composition shown in 2；The present invention is the molecular labeling primer developed by InDel sites, has the advantages of stability is good, and codominance is with being easily detected, can be used in large-scale molecular marker assisted selection breeding.
Description
Technical field
The present invention relates to agricultural biological technical field, particularly a kind of powdery mildew resistance gene in wheatStpk-VLabeled primer
And application.
Background technology
Wheat powdery mildew（Blumeria graminis f. sp. tritici）It is that harm is on the rise in Wheat Production
One of disease, after the eighties in 20th century, powdery mildew gradually rises to main disease in Chinese main Winter Wheat Area from Minor diseases
Evil, and occur throughout the year, 2004-2009 powdery mildews are 6,850,000 hm in the average occurring area in the whole nation 2, in Northern Winter area and Huang
Huaihe River Winter Wheat Area, powdery mildew are most important wheat diseases, and in the middle and lower reach of Yangtze River and the southwestern area of wheat, powdery mildew is to be only second to head blight
Or the second major disease of stripe rust.
At present, more than 50 a powdery mildew resistance genes, wherein powdery mildew resistance gene are found on 43 sites of wheatPm21
And highly resistance immune to the performance of most powdery mildew biological strains（Qi Lili etc., wheat powdery mildew new resistance source-genePm21Make
Thing journal, 1995,21（3）：257-262）, it is that the important gene resource that wheat breed is improved, Chen etc. utilizes ionising radiation side
Method obtains Wheat-Haynaldia villosa translocation lines 6VS/6AL, powdery mildew resistance genePm21It is positioned on 6V the short arm of a chromosome
（Chen PD, Qi LL, Zhou B, Zhang SZ, Liu DJ. Development and molecular
cytogenetic analysis of wheat-Haynaldia villosa 6VS/6AV translocation lines
specifying resistance to powdery mildew. Theor. Appl. Genet., 1995, 91: 1125-
1128）, in order to further clonePm21Gene, using biochip technology, is cloned into one and powdery mildew from haynaldia villosa 6VS
The relevant gene of resistanceStpk-V, which isPm21One important candidate gene of gene（Cao A, Xing L, Wang
X, Yang X, Wang W, Sun Y, Qian C, Ni J, Chen Y, Liu D, Wang X, Chen P.
Serine/threonine kinase gene Stpk-V, a key member of powdery mildew
resistance gene Pm21, confers powdery mildew resistance in wheat. Proc. Natl. Acad. Sci. USA, 2011, 108(19): 7727-7732）, using containingStpk-VThe 6VS translocation lines of gene,
It is bred as southern agriculture 9918, raises 15 wheat breeds such as wheat 18, the Resistant expression of the powdery mildew of these kinds is high anti-immune（Chen
PD, You CF, Hu Y, Chen SW, Zhou B, Cao AZ, Wang XE. Radiation-induced
translocations with reduced Haynaldiavillosa chromatin at the Pm21 locus for
powdery mildew resistance in wheat. Molecular Breeding, 2013, 31:477-484；Zhang Bai
Bridge, wheat 18 is raised using Rolling convergent backcross selection and breeding powdery-mildew-resistance wheat new varieties, and Chinese agronomy is circulated a notice of, and 2009,25（13）：74-77）.
At present, some existing scholars, which have screened, can be used to identifyPm21The molecular labeling of gene, such as OPH171900（Qi LL,
Cao MS, Chen PD, Li WL, Liu DJ. Identification, mapping and application of
polymorphic DNA associated with resistance gene Pm21 of wheat. Genome,1996,
39: 191-197）, OPH171400、SCAR1265、SCAR1400（Liu Zhi is brave etc., powdery mildew resistance gene in wheatPm21Molecular Identification
With marker assisted selection Acta Genetica Sinicas, 1999,26（6）：673-682；Liu Z, Sun Q, Yang T. Development
of SCAR markers linked to the Pm21 gene conferring resistance to powdery
mildew in common wheat. Plant breeding 1999, 118, 215-219）, but in the sieve of these marks
During choosing, researcher only analyzes the polymorphism between the material containing 6V chromosomes and common wheat, does not study these
The amplification situation in other 6 H. villosa chromosome materials are related to is marked, then research is found, molecular labeling OPH171900With
OPH171400Involving in haynaldia villosa other chromosomes（Beyond 6V chromosomes）Material in also amplify object tape, illustrate the two
Molecular labeling is not the mark that H. villosa chromosome 6VS specially changes；The primer that Cao Aizhong etc. is designed using probe Contig17515
（NAU/xibao15F and NAU/xibao15R）PCR amplification is carried out, can be from 6AS, 6BS, 6DS of common wheat and haynaldia villosa
PCR bands of different sizes are amplified on 6VS, thus develop a kind of based on PCR withPm21Chain co-dominant molecular mark
Remember NAU/xibao15902（Cao AZ, Wang XE, Chen YP, Zou XW, Chen PD. A sequene-specific
PCR marker linked with Pm21 distinguishes chromosomes 6AS, 6BS, 6DS ofTriticum aestivum and 6VS of Haynaldia villosa. Plant Breeding, 2006, 125:
201-205）, the mark containingPm21In the 6VS translocation lines of gene, the specific band of 902bp can be amplified, is haynaldia villosa
The mark that chromosome 6VS specially changes, but the mark amplifies 5 and 4 bands respectively in anti-, sense powdery mildew plant, during electrophoresis
It is not easily distinguishable.
InDel molecular labelings are to detect to be inserted into or lack in DNA section to find the inspection chain with target gene by PCR
Survey means, InDel are widely present in different plant individuals, as the genome sequencing of the important crops in part is completed and the
The generally use of two generation sequencing technologies, using improved bioinformatics method, can excavate a large amount of in different genotype
The InDel that can be used for developer molecule mark, average each gene is near or within there are an InDel site in theory,
These InDel can be located at intergenic region, can also be located at the promoter region of gene, and exon 1 includes sub-district and 3 ', and 5 '
UTR areas（Zhang Tifu etc., corn feature Insertion/Deletiion（InDel）The excavation of molecular labeling and its in cenospecies
Application Maize Sciences in Purity, 2012,20（2）：64-68）, but InDel is applied toStpk-VGenetic test, mesh
Before have not yet to see report.
The content of the invention
For problem present in current above-mentioned wheat powdery mildew Resistance detecting, there is provided a kind of labeled primer, for detecting
Powdery mildew resistance gene in wheatStpk-V,What the present invention was realized in：
A kind of and powdery mildew resistance gene in wheatStpk-VChain labeled primer, molecular labeling InDelw190It is by nucleotide
Sequence such as SEQ ID NO:Sense primer and such as SEQ ID NO shown in 1：What the anti-sense primer shown in 2 formed.
In the present invention, described and powdery mildew resistance gene in wheatStpk-VChain labeled primer is in resist powdery mildew of wheatStpk-VApplication in genetic test.
Application of the present invention, is to be detected material DNA as template, with SEQ ID NO:1 and SEQ ID NO：2 are
Primer carries out PCR amplification, and whether amplified production carries out electrophoresis detection containing the specific band that size is 190bp, and such as existing should
Band, then haveStpk-VGene.
In heretofore described application, PCR amplification is：PCR reaction systems are 25 μ L：DNA profiling 40 ng, primer SEQ
ID NO:1 and SEQ ID NO:2 each 5 pmol, 1 × PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP, Taq DNA
polymerase 1U；PCR reaction cycle programs are：94 DEG C of pre-degeneration 3min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C are prolonged
Stretch 30s, 35 circulations；72 DEG C of extension 10min；The electrophoresis detection detection refers to：After 1% Metaphor agarose gel electrophoresis
Detection, electrophoretic voltage 100v, electrophoresis time 2h.
The present invention is by rightStpk-VComplete genome sequence and Common Wheat Varieties China spring part sequencing sequence carry out
Analysis, findsStpk-VAt the 2572bp of gene order, there are the small fragment InDel of a 9bp to insert for China spring homologous sequence
Enter, pair of marks primer I nDel is devised on the InDel flanking sequencesw190, distinguished by PCR amplification and gel electrophoresisStpk-VAnd wheatStpkHomologous sequence site, contains common wheatStpkSite can only amplify 199bp bands, and containStpk-VThe wheat of gene can amplify two bands of 190bp and 199bp, therefore labeled primer InDelw190ForStpk-VGene
Special chemoattractant molecule labeled primer.
InDel labeled primers of the present invention, its amplified production are exactlyStpk-VGene sequence itself, can be directly to this
Assisting sifting is marked in gene, which carries out PCR expansions to common wheat or Triticum aestivum-Haynaldia villosa 6VS translocation lines
Increase, its product only has 1（190bp）Band or 2（190bp and 199bp）Band, easily distinguishable mildew-resistance plant and sense are white
Powder disease plant；And the molecular labeling developed by InDel sites has that stability is good, codominance and easily it is detected excellent
Point, can be used in large-scale molecular marker assisted selection breeding.
Brief description of the drawings
Fig. 1 is InDel in embodiment 1w190Mark PCR amplified production electrophoresis patterns；
Fig. 2 is NAU/xibao15 in embodiment 1902Mark PCR amplified production electrophoresis patterns；
Fig. 3 is that primer I nDel is marked in embodiment 2w190PCR is expanded in Triticum aestivum-Haynaldia villosa 6V alien chromosome lines
Product electrophoresis pattern；
Fig. 4 is InDel in embodiment 3w190Mark hybridizes F in peaceful wheat No. 9 with YC72-22For PCR amplified productions in plant
Electrophoresis pattern.
Embodiment
The labeled primer InDel that embodiment is related tow190：
Sense primer SEQ ID NO:1 5’–ACACGGGTTGCAGGAACATTG-3’；
Anti-sense primer SEQ ID NO:2：5’-AGCTTCTGAAGGCACACAGTAC-3’.
Embodiment is related to wheat source：
Common wheat China spring, feels powdery mildew（Liu steps on ability etc., the breeding improvement China agriculture of wheat China spring genetic background
Industry science, 2003,36（11）：1383-1389；Chen PD, You CF, Hu Y, Chen SW, Zhou B, Cao AZ,
Wang XE. Radiation-induced translocations with reduced Haynaldia villosa
chromatin at the Pm21 locus for powdery mildew resistance in wheat. Molecular
Breeding, 2013, 31:477-484）；
Peaceful wheat No. 9, middle sense powdery mildew（Yao Jinbao etc., the research and utilization of fine wheat parent Ning Mai 9, nuclear agricultural science report,
2012,26（1）：17-21）；
Triticum aestivum-Haynaldia villosa 6VS/6AL translocation line 92R137, mildew-resistance（Qi Lili etc., wheat powdery mildew newly resists
Source-genePm21Acta Agronomica Sinica, 1995,21（3）：257-262；Chen PD, Qi LL, Zhou B, Zhang SZ,
Liu DJ. Development and molecular cytogenetic analysis of wheat-Haynaldia villosa 6VS/6AV translocation lines specifying resistance to powdery mildew.Theor. Appl. Genet., 1995, 91: 1125-1128）；
Triticum aestivum-Haynaldia villosa 6VS Small piece transpositions system YC72-2, mildew-resistance（Yang XM, Cao AZ, Sun
YL, Chen PD. Tracing the location of powdery mildew resistance-related geneStpk-V by FISH with a TAC clone in Triticum aestivum–Haynaldia villosa alien
chromosome lines. Chinese Science Bulletin, 2013, 58(33):4084-4091）；
Southern agriculture 9918（9918. Agricultural University Of Nanjing's journal of Chen Peidu etc., mildew-resistance High-yield Wheat new varieties Nan Nong,
2002,25（4）：105-106）；
Raise wheat 18（Zhang Baiqiao application Rolling convergent backcross selection and breeding powdery-mildew-resistance wheat new varieties are raised the Chinese agronomy of wheat 18. and are led to
Report, 2009,25（13）：74-77）；
Zheng wheat 883（Mulberry main forces etc., Molecular Identification and the molecular labeling auxiliary of Wheat in Henan Province kind powdery mildew resistance gene
Breeding North China agronomy report, 2006,21（1）：86-91；）；
Triticum aestivum-Haynaldia villosa 6V addition lines 06R33,6V substitution line 06R41,6V addition Deletion line del6VS-1,6VS is easy
Position is 92R139,92R90,6VS Small piece transpositions system YC138-4, NJ4, YC24, NJ1-15, YC146-4（Yang XM, Cao
AZ, Sun YL, Chen PD. Tracing the location of powdery mildew resistance-
related gene Stpk-V by FISH with a TAC clone in Triticum aestivum–Haynaldia villosa alien chromosome lines. Chinese Science Bulletin, 2013, 58(33):4084-
4091；The bright of Yang Xue and wheat anti-powdery mildew are relevantHv-S/TPKThe physical positioning of gene and its Transgenic plant of wheat
FISH identification Agricultural University Of Nanjing Ph.D. Dissertations, 2012）.
1 labeled primer InDel of embodimentw190With NAU/xibao15902Contrast in different wheat genetic materials is real
Test
Respectively with common wheat China spring, peaceful wheat No. 9, Triticum aestivum-Haynaldia villosa 6VS/6AL translocation lines 92R137, common
The DNA of wheat-haynaldia villosa 6VS Small piece transpositions system YC72-2 is template, with InDelw190PCR amplification is carried out for primer.
PCR reaction systems are 25 μ L, include 40 ng of DNA profiling, SEQ ID NO：1 and SEQ ID NO:2 primers each 5
Pmol, 1 × PCR buffer, 1.5 mM MgCl2, 0.2 mmol dNTP, Taq DNA polymerase 1U；
PCR reaction cycle programs are：94 DEG C of pre-degenerations 3min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s,
35 circulations；72 DEG C of extension 10min；4 DEG C of preservations；
1% Metaphor agarose gel electrophoresis of PCR product, electrophoretic voltage 100v, electrophoresis time 2h, as a result such as Fig. 1
It is shown, wherein, swimming lane M is molecular weight marker, and swimming lane 1 is common wheat China spring, and swimming lane 2 is peaceful wheat No. 9, and swimming lane 3 is common
Wheat-haynaldia villosa 6VS/6AL translocation line 92R137, swimming lane 4 is Triticum aestivum-Haynaldia villosa 6VS Small piece transpositions system YC72-2, general
Logical wheat-haynaldia villosa 6VS/6AL translocation lines 92R137 and Triticum aestivum-Haynaldia villosa 6VS small fragment is easily that can expand in YC72-2
Increase and specific band and 199bp band of the size for 190bp, and only amplified in common wheat China spring and Ning Mai 9
The band of 199bp.
With common wheat China spring, peaceful wheat No. 9, Triticum aestivum-Haynaldia villosa 6VS/6AL translocation lines 92R137, common wheat-
The DNA of haynaldia villosa 6VS Small piece transpositions system YC72-2 is template, NAU/xibao15902For primer, PCR amplification, primer are carried out
Sequence and amplification method are such as《A sequene-specific PCR marker linked withPm21 distinguishes
chromosomes 6AS, 6BS, 6DS of Triticum aestivum and 6VS of Haynaldia villosa.
》（Cao AZ, Wang XE, Chen YP, Zou XW, Chen PD.Plant Breeding, 2006, 125: 201-
205）Described, for its amplification as shown in Fig. 2, wherein, swimming lane M is molecular weight marker, swimming lane 5 is common wheat China spring, swimming
Road 6 is peaceful wheat No. 9, and swimming lane 7 is Triticum aestivum-Haynaldia villosa 6VS/6AL translocation line 92R137, and swimming lane 8 is Triticum aestivum-Haynaldia villosa
6VS Small piece transpositions system YC72-2, mildew-resistance plant Triticum aestivum-Haynaldia villosa 6VS/6AL translocation lines 92R137 with it is common small
Wheat-haynaldia villosa 6VS Small piece transpositions system YC72-2 amplifies 5 bands, and susceptible powdery mildew plant common wheat China spring
4 bands are amplified with peaceful wheat No. 9.
2 labeled primer InDel of embodimentw190Analysis in Triticum aestivum-Haynaldia villosa 6V alien chromosome lines
Using the DNA of different wheat lines as template, InDelw190For primer, to Triticum aestivum-Haynaldia villosa 6V heterochromosomes
It is that wheat carries out PCR amplification and electrophoresis, PCR reaction systems and electrophoresis are same as Example 1, and electrophoresis result is as shown in figure 3, wherein
Swimming lane M is molecular weight marker, and swimming lane 1 is Triticum aestivum-Haynaldia villosa 6VS/6AL translocation line 92R137, and swimming lane 2 is in common wheat
State's spring, swimming lane 3 are southern agriculture 9918, and swimming lane 4 is raises wheat 18, and swimming lane 5 is Zheng wheat 883, and swimming lane, 6 be 06R33, and swimming lane 7 is 06R41,
Swimming lane 8 is del6VS-1, and swimming lane 9 is 92R139, and swimming lane 10 is 92R90, and swimming lane 11 is YC138-4, and swimming lane 12 is NJ4, swimming lane
13 be YC24, and swimming lane 14 is NJ1-15, and swimming lane 15 is YC146-4；The result shows that in mildew-resistance plant Triticum aestivum-Haynaldia villosa
What can be stablized in 6V alien chromosome lines amplifies the specific band and 199bp bands that size is 190bp, and susceptible white
Powder disease kind common wheat China spring can only amplify 199bp bands.
Embodiment 3
Labeled primer InDelw190Hybridize F with wheat-haynaldia villosa 6VS Small piece transpositions system YC72-2 in peaceful wheat No. 92Dai Zhi
Verified in strain
With the F of peaceful No. 9 × YC72-2 of wheat2For 18 plant in colony and the DNA of 2 parents PCR is carried out for template
Amplification, PCR reaction systems and electrophoresis are same as Example 1, and electrophoresis result is as shown in figure 4, wherein M is molecular weight marker, swimming lane 1
For parent YC72-2, swimming lane 2 is parent Ning Mai 9, and swimming lane 3-20 is F2For plant, swimming lane 5,6,10,11,13,14,15,16,
17th, 18,20 F2For amplified in the parent YC72-2 of plant and swimming lane 1 size be 190bp specific band and
199bp bands, are mildew-resistance plant, the F of swimming lane 3,4,7,8,9,12,192For single plant and the parent Ning Mai 9 of swimming lane 2
199bp bands are only amplified, to feel powdery mildew plant.
SEQUENCE LISTING
<110>Jiangsu Province Agriculture Science Institute
<120>The labeled primer of powdery mildew resistance gene in wheat Stpk-V a kind of and application
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 1
acacgggttg caggaacatt g 21
<210> 2
<211> 22
<212> DNA
<213>It is artificial synthesized
<400> 2
agcttctgaa ggcacacagt ac 22
Claims (3)
- A kind of 1. powdery mildew resistance gene in wheatStpk-VLabeled primer, it is characterised in that powdery mildew resistance gene in wheatStpk-V Labeled primer InDelw190, by nucleotide sequence such as SEQ ID NO:Sense primer and such as SEQ ID NO shown in 1：Shown in 2 Anti-sense primer composition.
- 2. powdery mildew resistance gene in wheat as claimed in claim 1Stpk-VLabeled primer in resist powdery mildew of wheatStpk-VGene Application in detection；The application refers to, using the DNA of detected wheat plant as template, with SEQ ID NO:1 and SEQ ID NO：2 be primer PCR amplification is carried out, whether amplified production carries out electrophoresis detection containing the specific band that size is 190bp, if in the presence of having Powdery mildew resistance gene in wheatStpk-V, for anti-light flour disease plant.
- 3. application as claimed in claim 2, it is characterised in that the PCR amplification is：PCR reaction systems are 25 μ L：40 ng of DNA profiling, primer SEQ ID NO:1 and SEQ ID NO:2 each 5 pmol, 1 × PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP, Taq DNA polymerase 1U；PCR reaction cycle programs are：94 DEG C of pre-degeneration 3min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s, 35 Circulation；72 DEG C of extension 10min；The electrophoresis detection detection refers to：Detected after 1% Metaphor agarose gel electrophoresis, electrophoretic voltage 100v, during electrophoresis Between 2h.
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| CN108546776B (en) * | 2018-07-16 | 2020-03-31 | 四川农业大学 | InDel marker of poland dwarf wheat, identification method and application |
| CN109355426B (en) * | 2018-12-17 | 2021-10-15 | 山西省农业科学院作物科学研究所 | Diagnostic molecular marker for detecting powdery mildew resistance gene Pm2a of wheat and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1661106A (en) * | 2004-12-30 | 2005-08-31 | 南京农业大学 | Tagged primer chained to Pm21 gene of anti powdery mildew of wheat and application |
| CN1737151A (en) * | 2005-07-12 | 2006-02-22 | 南京农业大学 | Wheat anti-powdery mildew gene Pm21 linked codorminant PCR marker and its usage |
| CN101121945A (en) * | 2007-06-29 | 2008-02-13 | 天津师范大学 | Special RAPD molecule marker for wheat 6VS |
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2014
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1661106A (en) * | 2004-12-30 | 2005-08-31 | 南京农业大学 | Tagged primer chained to Pm21 gene of anti powdery mildew of wheat and application |
| CN1737151A (en) * | 2005-07-12 | 2006-02-22 | 南京农业大学 | Wheat anti-powdery mildew gene Pm21 linked codorminant PCR marker and its usage |
| CN101121945A (en) * | 2007-06-29 | 2008-02-13 | 天津师范大学 | Special RAPD molecule marker for wheat 6VS |
Non-Patent Citations (2)
| Title |
|---|
| A sequene-specific PCR marker linked with Pm21 distinguishes chromosomes 6AS, 6BS, 6DS of Triticum aestivum and 6VS of Haynaldia villosa;Cao AZ et al.;《Plant Breeding》;20061231(第125期);201-205 * |
| Serine/threonine kinase gene Stpk-V, a key member of powdery mildew resistance gene Pm21, confers powdery mildew resistance in wheat;Cao A et al.;《Proc. Natl. Acad. Sci》;20110510;第108卷(第19期);7727-7732 * |
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