CN104975073A - Application of specific primers for identifying physiological race THTS of puccinia triticina - Google Patents
Application of specific primers for identifying physiological race THTS of puccinia triticina Download PDFInfo
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- CN104975073A CN104975073A CN201410139689.6A CN201410139689A CN104975073A CN 104975073 A CN104975073 A CN 104975073A CN 201410139689 A CN201410139689 A CN 201410139689A CN 104975073 A CN104975073 A CN 104975073A
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- 241001246061 Puccinia triticina Species 0.000 title abstract description 4
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 10
- 238000012408 PCR amplification Methods 0.000 claims description 9
- 241000221300 Puccinia Species 0.000 claims description 9
- 238000012797 qualification Methods 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 5
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- 229910052709 silver Inorganic materials 0.000 claims description 5
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- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000012498 ultrapure water Substances 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- 239000003147 molecular marker Substances 0.000 abstract description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract 2
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- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a primer application for identifying puccinia triticina physiological race THTS and a THTS race specific DNA sequence, wherein the primer sequence is a primer pair consisting of upstream and downstream nucleotide sequences of a primer Ptssr0125 in a sequence table. The EST-SSR molecular marker can be used for quickly identifying physiological races of the strains of the puccinia triticina.
Description
Technical field
The present invention relates to a kind of primer sequence for the identification of tritci and application thereof.
Background technology
By puccinia triticinia (
puccinia triticina) wheat leaf rust that causes is one of disease of generally occurring of world Mai Qu, its Occurrence & epidemic makes yield and quality of wheat be had a strong impact on, and being affects the significant obstacle that world wheat produces.Because wheat breed is different with occurrent time, 7%-30% can be caused, even more than 50% production loss (Huerta-Espino et al. 2011).Facts have proved, control the application that this disease is most economical, safety, effective ways are disease-resistant varieties.But the forfeiture of the anti-leaf rust property of kind is serious problems of disease-resistant variety application, and its major cause is the generation of leaf rust variation and new toxicity microspecies.Therefore, tritci monitoring and epidemics forecast have become control wheat leaf rust, have instructed the important step of disease-resistant wheat breed breeding and layout.But puccinia triticinia is a kind of obligate parasite, cannot artificial culture be carried out, and the general survey of physiological strain and monitoring method numerous and diverse, time-consuming, accuracy is also vulnerable to the impact of qualification condition, therefore, a kind of easy, quick, novel method that accuracy is high need be found.
At present, DNA marker technology has been widely used in fungal population's biology, epidemiology research.RAPD, RFLP, AFLP and SSR molecular marker have been widely used in the research of puccinia triticinia population genetic variations.Wherein SSR marker because its polymorphism is high, codominance, good stability and be widely applied, but the specific molecule marker of tritci is also little at present, and DNA molecular marker cannot be utilized to carry out Race identification fast.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of primer sequence for the identification of tritci, for Rapid identification tritci type to be measured.
For solving the problems of the technologies described above, primer sequence of the present invention is Ptssr0125, is made up of, table 1 sequence table middle and upper reaches sequence (20 bases) and downstream sequence (20 bases).
Table 1 is for detecting two primer upstream and downstream sequences of leaf rust resistance gene
| Primer | Upstream sequence | Downstream sequence |
| Ptssr0125 | 5' ATCGTGTCATGCAACCAAAA 3' | 5' AGAGAGGGACGTGAGGGATA 3' |
Molecule marker for the identification of tritci provided by the present invention is Ptssr0125, by primer Ptssr0125 amplification acquisition 200 bp and 178bp band.
Present invention also offers the application of primer sequence in screening tritci.
The concrete grammar of the present invention's application is: with puccinia triticinia genomic dna to be measured for template, utilize primer Ptssr0125 to carry out pcr amplification, then detect amplified production; When amplified production has 200bp and 178bp band simultaneously, then the physiological strain type of wheat leaf rust bacterial strain to be measured is THTS.
Pcr amplification described in the present invention's application:
20 μ l PCR reaction systems are: template DNA 60 ng, Taq enzyme 0.5 U, upstream and downstream primer (5 μm of olL
-1) each 0.4 μ L, dNTP(10 mmolL
-1) 0.4 μ L, 10 × PCR damping fluid 2 μ L, with aseptic ultrapure water postreaction system to 20 μ L;
PCR response procedures is: first 94 DEG C of 5 min; Then 94 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 1 min, totally 35 circulations; Last 72 DEG C of 5 min, 4 DEG C of preservations.
Detection is carried out to amplified production be described in the present invention's application: to amplified production electrophoretic separation in the non-denaturing polyacrylamide gel of 10% (w/v), the then colour developing of silver dye.
The beneficial effect that produces of technique scheme is adopted to be: the EST-SSR specific mark that the invention provides a new tritci THTS, utilize this mark can Rapid identification, monitoring physiological strain THTS, thus lay the foundation for setting up the molecular monitoring technical system with the Wheat in China leaf rust physiological strain of practical value.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
Fig. 1 is to the pcr amplification result of 14 physiological strains, 21 bacterial strains with EST-SSR primer Ptssr0125.In each swimming lane, M represents molecular weight standard, and 1-21 represents 21 in 213 wheat leaf rust bacterial strains, wherein 11 and 12 bacterial strains are THTS physiological strain, and 2 and 19 bacterial strains are THTT physiological strain, 3,9,15 and 18 bacterial strains are THST physiological strain, and other swimming lane respectively represents a kind of physiological strain.
Embodiment
For the identification of the primer of tritci THTS, the primer pair be made up of the upstream and downstream nucleotide sequence of primer Ptssr0125 in sequence table.
The concrete grammar applying this primer sequence qualification tritci THTS is as follows:
1, extracting Wheat volatiles DNA to be measured is template;
2, take Ptssr0125 as primer, puccinia triticinia genomic dna to be measured is template, carries out pcr amplification.20 μ l PCR reaction systems are: template DNA 60 ng, Taq enzyme 0.5 U, upstream and downstream primer (5 μm of olL
-1) each 0.4 μ L, dNTP(10 mmolL
-1) 0.4 μ L, 10 × PCR damping fluid 2 μ L, with aseptic ultrapure water postreaction system to 20 μ L; Response procedures is 94 DEG C of denaturation 5min, carries out 35 circulations subsequently: 94 DEG C of sex change 30 s, 55 DEG C of annealing 30 s, and 72 DEG C extend 1 min; 72 DEG C extend 5 min, last 4 DEG C of preservations.
3, separation detection is carried out to amplified production: in amplified production, add non denatured load sample indicator, it is electrophoretic separation in the non-denaturing polyacrylamide gel of 10% (w/v) in concentration, the colour developing of silver dye, if the band containing 200bp and 178bp size in amplified production, then the Pathogenic Types of puccinia triticinia to be measured is THTS.Described non denatured load sample indicator consists of: 0.25% (w/v) tetrabromophenol sulfonphthalein, and 0.25% (w/v) diformazan cyanophenyl and 40% (w/v) sucrose, solvent is distilled water.
In following embodiment, method therefor is ordinary method if no special instructions, and all primer synthesis complete by Hua Da Genetic Biotechnologies company limited.
Experimental example:
Tritci THTS, EST-SSR mark the acquisition of Ptssr0125:
Select 21 pairs of EST-SSR primers, all primer sequences are derived from Wang
et al.the puccinia triticinia EST-SSR primer of exploitation in 2010, with the genomic dna of 213 wheat leaf rust bacteria strains for template, carry out PCR detection, 20 μ L PCR reaction systems are: template DNA 60 ng, Taq enzyme 0.5 U, upstream and downstream primer (5 μm of olL
-1) each 0.4 μ L, dNTP(10 mmolL
-1) 0.4 μ L, 10 × PCR damping fluid 2 μ L, with aseptic ultrapure water postreaction system to 20 μ L.PCR response procedures is: first 94 DEG C of 5 min; Then 94 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 1 min, totally 35 circulations; Last 72 DEG C of 5 min, 4 DEG C of preservations.
Then as follows pcr amplification product is carried out to the native polyacrylamide gel electrophoresis detection of 10% (w/v): get 20 μ L amplified productions, add 4 μ L non denatured load sample indicator (0.25% (w/v) tetrabromophenol sulfonphthaleins, 0.25% (w/v) diformazan cyanophenyl and 40% (w/v) sucrose), 3 μ L got by every part of amplification sample is electrophoretic separation in the non-denaturing polyacrylamide gel of 10% in concentration
The colour developing of silver dye.14 EST-SSR sites Ptssr0083, Ptssr0019, Ptssr5649, Ptssr0085, Ptssr2948, Ptssr0243, Ptssr0125, Ptssr5594, Ptssr0189, Ptssr0481, Ptssr0639, Ptssr6542, Ptssr0182 and Ptssr6386 have polymorphism between 213 bacterial strains as a result, wherein primer Ptssr0125 detected two kinds of electrophoresis banding patterns, divide and be called banding pattern A and B, all bacterial strains all can amplify the DNA fragmentation of 200bp, see banding pattern A in Fig. 1, physiological strain THTS also can amplify the DNA fragmentation of 178bp, sees banding pattern B in Fig. 1.
Embodiment 1:
1, the genomic dna extracting 213 leaf rust strains is template;
2, take Ptssr0125 as primer, 213 leaf rust genomic dnas are template, carries out pcr amplification.20 μ l PCR reaction systems are: template DNA 60 ng, Taq enzyme 0.5 U, downstream primer (5 μm of olL
-1) each 0.4 μ L, dNTP(10 mmolL
-1) 0.4 μ L, 10 × PCR damping fluid 2 μ L, with aseptic ultrapure water postreaction system to 20 μ L.Response procedures is: first 94 DEG C of 5 min; Then 35 circulations: 94 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 1 min; Last 72 DEG C of 5 min, 4 DEG C of preservations.
3, separation detection is carried out to amplified production: in amplified production, add non denatured load sample indicator, then be electrophoretic separation in the denaturing polyacrylamide gel of 10% (w/v) in concentration, the colour developing of silver dye, in the pcr amplification collection of illustrative plates that polyacrylamide gel electrophoresis detects, that have 200bp and 178bp size strip is physiological strain THTS simultaneously, and what only have 200bp size strip is other physiological strain.
The distinguished sequence of THTS microspecies, namely B band (178bp) is as follows: ATCGTGTCATGCAACCAAAAAGAACAAGGATGATGATGATGATGATGATGATGAGA GGGGAGAAAAAAAGGCAAGGAAAAAAAAAGATGATGAATAAGACAAAAAGAACCTA GGTATTGGAAAAAATCTAGTAAAATGAATAGAAAGAGAACAAGAAATATCCCTCAC GTCCCTCTCT
Claims (5)
1. for the identification of a primer sequence for tritci, the primer pair be made up of the upstream and downstream nucleotide sequence of primer Ptssr0125 in sequence table.
2. the application of primer sequence according to claim 1 in qualification tritci.
3. the application of primer sequence according to claim 2 in qualification tritci, is characterized in that: with puccinia triticinia strain gene group DNA to be measured for template, carry out pcr amplification, then detect amplified production with primer Ptssr0125; Amplified production contains 200 bp bands, and during containing 178 bp band, then the physiological strain type of puccinia triticinia to be measured is THTS.
4. the application of primer sequence according to claim 3 in qualification tritci, is characterized in that, described pcr amplification:
20 μ L PCR reaction systems are: template DNA 60 ng, Taq enzyme 0.5 U, upstream and downstream primer (5 μm of olL
-1) each 0.4 μ L, dNTP(10 mmolL
-1) 0.4 μ L, 10 × PCR damping fluid 2 μ L, with aseptic ultrapure water postreaction system to 20 μ L;
PCR response procedures is: first 94 DEG C of 5 min; Then 94 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 1 min, totally 35 circulations; Last 72 DEG C of 5 min, 4 DEG C of preservations.
5. the application of the primer sequence according to claim 3 or 4 in qualification tritci, it is characterized in that, described detection is carried out to amplified production be: to amplified production electrophoretic separation in the non-denaturing polyacrylamide gel of 10% (w/v), the colour developing of silver dye.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410139689.6A CN104975073A (en) | 2014-04-09 | 2014-04-09 | Application of specific primers for identifying physiological race THTS of puccinia triticina |
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| CN201410139689.6A CN104975073A (en) | 2014-04-09 | 2014-04-09 | Application of specific primers for identifying physiological race THTS of puccinia triticina |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058602A (en) * | 2017-06-26 | 2017-08-18 | 河北农业大学 | Primer group of wheat puccinia triticina EST-SSR molecular marker and detection method and application thereof |
| CN110205400A (en) * | 2019-06-27 | 2019-09-06 | 河北农业大学 | Kit and method for detecting puccinia triticina |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101967518A (en) * | 2010-05-26 | 2011-02-09 | 河北农业大学 | Method for screening wheat leaf rust resistance gene Lr24 and special primer thereof |
-
2014
- 2014-04-09 CN CN201410139689.6A patent/CN104975073A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101967518A (en) * | 2010-05-26 | 2011-02-09 | 河北农业大学 | Method for screening wheat leaf rust resistance gene Lr24 and special primer thereof |
Non-Patent Citations (3)
| Title |
|---|
| XIBEN WANG ET.AL: "Development of est-derived simple sequence repeat markers for wheat leaf rus fungus,puccinia triticina eriks.", 《CAN.J.PLANT PATHOL.》 * |
| 李伟,黄彬主编: "《分子诊断学》", 30 September 2004, 北京：中国医药科技出版社 * |
| 赵盼盼等: "中国冀、鲁、川新小麦叶锈菌群体est-ssr遗传多样性分析", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058602A (en) * | 2017-06-26 | 2017-08-18 | 河北农业大学 | Primer group of wheat puccinia triticina EST-SSR molecular marker and detection method and application thereof |
| CN107058602B (en) * | 2017-06-26 | 2020-01-21 | 河北农业大学 | Primer group of wheat puccinia triticina EST-SSR molecular marker and detection method and application thereof |
| CN110205400A (en) * | 2019-06-27 | 2019-09-06 | 河北农业大学 | Kit and method for detecting puccinia triticina |
| CN110205400B (en) * | 2019-06-27 | 2022-08-02 | 河北农业大学 | Kit and method for detecting puccinia triticina |
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