CN106148510A - Resistance gene of rice blast Pi5 specific Function molecular marker and application thereof - Google Patents
Resistance gene of rice blast Pi5 specific Function molecular marker and application thereof Download PDFInfo
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- CN106148510A CN106148510A CN201610431981.4A CN201610431981A CN106148510A CN 106148510 A CN106148510 A CN 106148510A CN 201610431981 A CN201610431981 A CN 201610431981A CN 106148510 A CN106148510 A CN 106148510A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
Resistance gene of rice blast Pi5 specific Function molecular marker and application thereof, named Pi5InDel, nucleotide sequence is as shown in SEQ ID NO.1.Rice blast resistance gene Pi5 gene specific molecular marker specificity provided by the present invention is high；Low cost, high flux in actual applications；The rice germplasm containing rice blast resistance gene Pi with screening can be widely used in the colony that genetic background is different.Utilize this molecular marker can improve this gene in molecular marker assisted selection breeding, gene pyramiding breeding, and the efficiency utilized in transgenic breeding.
Description
Technical field
The present invention relates to agricultural biological technical field, more particularly to rice blast technical field, particularly to a kind of rice blast
Sick resistant gene Pi5 specific Function molecular marker and application thereof.
Background technology
Oryza sativa L. is one of the most most important cereal crops, and the economic loss caused because of rice blast every year reaches billions of U.S.
Unit (Zheng Zhao etc., 2009).China Oryza sativa L. main producing region is nearly all endangered by rice blast, and year, onset area was to 330-570 ten thousand public affairs
Hectare, lose several hundred million kilograms of Oryza glutinosa.From the perspective of grain security and Genetic and breeding in rice, cultivating disease-resistant variety is preventing and treating rice blast
One of sick most economical effective method.But owing to rice blast biological strain pathogenicity variation is fast, the resistance meeting of single resistant variety
Gradually lose (the abundant institute etc. that obtains, 2011)；And traditional resistant breeding strategy features such as to there is workload big, cycle length.Therefore, compel
It is essential and to change the strategy of tradition breeding for disease resistance.Based on molecular marker assisted selection (Marker-associated
Selection, MAS) the disease-resistant molecular breeding of technology has the selection to objective trait not by gene expression and environmental condition shadow
Ring, can carry out in any stage of plant strain growth, can segregating generation identify rapidly and accurately plant genotype, can also be effectively
Overcome the advantages such as the target gene Linkage drag with unfavorable gene, be used in the resistance breeding field of rice blast.But
Molecular marker closely linked with genes of interest is likely limited by genetic background in application process, in different colonies
Middle the polymorphism of parent need to be detected；It addition, have the molecular marker of certain distance to remain because of dye with genes of interest
Colour solid occurs exchange to lose the probability of genes of interest, and this makes to there will be wrong choosing or leakage in genes of interest qualification process
Choosing situation (Hayashi et al., 2006；Ingvardsen et al., 2008).But, based on gene internal sequence institute
The gene specific molecular marker of exploitation can make the problems referred to above effectively be solved, thus improves the utilization effect of resistant gene
Rate, contributes to carrying out of rice blast resistance breeding.
Pi5 is the wide spectrum blast resistant gene (Jeon etc., 1994) being positioned on the 9th chromosome, order-checking find Pi5-1 and
Pi5-2 has 5 and 6 exons respectively.The rice blast resistance of Pi5 mediation needs have the two gene simultaneously, resistant gene Pi5
The rice blast resistance of wide spectrum shown makes it have great using value in rice blast resistance breeding work.But, Pi5
Distribution situation in China's Rice Germplasm Resources and kind does not understands, constrains this resistant gene extensively should in breeding
With.In order to enable to be applied in Oryza sativa L. resistance breeding work by rice blast broad-spectrum resistance gene Pi5 more accurately and effectively, it is necessary to
The gene internal of resistant gene Pi5 develops InDel molecular marker special for Ρ i5, and this gene specific molecular marker can be right
The accuracy rate theoretical value of target gene screening reaches 100%.
Summary of the invention
Solve the technical problem that: in order to enable faster, more effectively rice blast resistance gene Pi5 is applied to resistance breeding
In, the present invention provides a kind of rice blast resistance gene Pi5 gene specific molecular marker Pi5InDel and application thereof.This molecule mark
Note Pi5InDel difference based on gene order insertion and deletion (Insertion-deletion, InDel) research obtains, and it can carry
This gene high is in molecular marker assisted selection breeding, gene pyramiding breeding, and the utilization ratio in transgenic breeding.
Technical scheme: resistance gene of rice blast Pi5 specific Function molecular marker, named Pi5InDel, nucleic acid
Sequence is as shown in SEQ ID NO.1.
The above-mentioned rice blast resistance kind carrying rice blast resistance gene Pi5 is resistant rice cultivars IRBL5-M.
The application in differentiating Rice Blast resistant gene of the above-mentioned molecular marker.
Above-mentioned application comprises the steps of: (1) expands: utilize primers F l and the Rl genome to rice varieties to be detected
Carry out PCR；(2) detection: molecular size range be 93bp nucleotide fragments, rice varieties the most to be detected carries rice blast
Resistant gene Pi5；If being not detected by the nucleotide fragments that molecular size range is 93bp, rice varieties the most to be detected is not taken
With rice blast resistance gene Pi5.
For expanding the primer pair of described molecular marker, nucleotide sequence is as follows:
SEQ ID NO.2:Fl:5 '-GTTTCGAGATAGTGCTAA-3 '；
SEQ ID NO.3:Rl:5 '-GAATGACAGTAATAGAAA-3 '.
A kind of test kit differentiating Rice Blast resistant gene, containing nucleotide sequence as shown in SEQ ID NO.1
Pi5InDel, and the primer pair as described in SEQ ID NO.2 and SEQ ID NO.3.
The principle of the present invention: insertion/deletion (insertion-deletion, InDel) labelling PCR-based amplification technique,
Because of its good stability, polymorphism is high, classification system is simple, starts to be applied to the genetic analysis of animals and plants colony, marker assisted selection
And the field such as medical diagnosis.The present invention finds the internal InDel occurred of Pi5 gene order by the method for Multiple Sequence Alignment,
The upstream and downstream design primer in site is to F1/R1 accordingly, carries out standard PCR amplification with it, and the amplification carrying Pi5 rice varieties is produced
Thing molecular size range is 93bp, when polyacrylamide gel electrophoresis of the presently claimed invention detects, carries Pi5 rice varieties
Digestion products molecular weight is 93bp, and does not carry the digestion products molecular weight of the rice varieties of Pi5 and present with 95bp or without band.
Beneficial effect: the molecular marker specificity that (1) present invention provides is high: the genome area at Pi5 place also exists merit
Energy nand function gene non-characteristic fragment, utilizes existing Marker Identification to have a lot of false-positive result and occurs.The present invention carries
The molecular marker of confession is to carry out sequence polymorphism seeking through constantly, and obtained by experimental verification, can be significantly by Pi5
Separate with the paralog gene regions being present in this genome area with Pi5 very high homology.
(2) the molecular marker low cost in actual applications of present invention offer, high flux: at present, InDel marker detection side
Formula is simple and convenient, requires relatively low to instrument and equipment and technology, can carry out on electrophoretic techniques platform.The present invention provides
The amplified production banding pattern of InDel molecular marker is clearly simple, and its stability and product separating effect are preferable；Due to amplified production relatively
Short, relatively low to DNA prescription, and sample size is required less, even can expand the DNA of height degraded, be particularly well-suited to raw
Produce in practice.
(3) present invention is first Pi5 gene specific InDel labelling developed for Pi5 gene internal sequence.This
Pi5 successfully can be separated with other the non-Pi5 gene regions being positioned on this site by invention by the method for electrophoresis detection.Up till now
Till, also there is no the report about this kind of labelling.Pi5 specific molecular marker Pi5InDel provided by the present invention is one and is total to
Dominant marker, in actual application, its reliability and accuracy are better than dominant marker.Present invention can apply to germ plasm resource
Analyze, Pi5 transgenic is identified, gene pyramiding (the especially allelic polymerization of sequence very high homology), and based on MAS skill
In the Oryza sativa L. resistance breeding work of art.This labelling is present in Pi5 gene internal, therefore to the screening capacity theoretical value of Pi5 up to
100%, such screening capacity is better than the molecular marker chain with Pi5 reported.
(4) molecular marker that the present invention provides can be widely used in the colony that genetic background is different.Existing and Pi5 connects
The molecular marker major part of lock is both for what the sequence polymorphism of same 2 different parents of colony was developed, these labellings
The suitability in other colony is limited.The present invention be applicable to the germplasms evaluation under any genetic background, transgenic breeding,
Gene pyramiding and resistance breeding based on MAS technology, it is not necessary to repeat the screening of parent's polymorphism, substantially increase breeding
Efficiency.
Therefore, rice blast resistance gene Pi5 gene specific molecular marker provided by the present invention has important application
It is worth, utilizes this labelling can improve this gene and educate at molecular marker assisted selection breeding, gene pyramiding breeding, and transgenic
The efficiency utilized in kind.
Accompanying drawing explanation
Fig. 1 is the amplification situation that JJ813 is marked in 12 parts of representative kinds, wherein: swimming lane M is DNA ladder, swimming
The DNA profiling in road 2～12 be followed successively by resistant variety IRBL5-M (Pi5), middle spend 16, open country, Guangdong accounts for, flower joint is glutinous, the rich B in sky, bright extensive 63,
Japan is fine, 9311, osmanthus is towards No. 2, even round-grained rice 7, II-32A, extensively account for 63s.
Fig. 2 is the contrast verification figure of Pi5 gene specific molecular marker Pi5SNP Yu JJ813.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention do not limit
In this.
Embodiment 1: rice blast resistance gene Pi5 gene specific molecular marker and design of primers thereof and detection
(1) analysis in Pi5 insertion/deletion (insertion-deletion, InDel) site:
Pi5 genome sequence (the Genebank number of the including difference published is downloaded from public database
For EU869185, EU869186), be designed primer for Pi5 site, examination Pi5 special, can be different from this site its
Insertion/deletion (InDel) the difference site of his rice blast resistance allele.There is the Oryza sativa L. rice blast of gene specific InDel
Sick resistant gene Pi5 is as follows with other sequence alignment result wide in variety containing non-Pi5 functional gene, wherein, and two of disappearance
Base portion is Pi5 gene insertion/deletion (InDel) the difference site identified.
IRBL5-M:TATATTTTTCTGTCCCTTAAAATAGTTTTTTTT--CTATTACTGTCAT TCATGTGACTCAT
TTTGTTTTTTAG
Zegu:TATATTTTTCTGTCCCTTAAAATAGTTTTTTTT--CTATTACTGTCATTCA TGTGACTCATTTT
GTTTTTTAG
Lianjing 7hao:TATATTTTTCTGTCCCTTAAAATAGTTTTTTTT--CTATTACTGTCATTCA TGTG
ACTCATTTTGTTTTTTAG
Lianjing11hao:TATATTTTTCTGTCCCTTAAAATAGTTTTTTTT--CTATTAC TGTCATTCATGTG
ACTCATTTTGTTTTTTAG
Yueyezhan:TATATTTTTCTGTCCCTTAAAATAGTTTTTTTTTTCTATTACTGTC ATTCATGTGACTC
ATTTTGTTTTTTAG
Zhonghua16:TATATTTTTCTGTCCCTTAAAATAGTTTTTTTTTTCTATTACTGT CATTCATGTGACT
CATTTTGTTTTTTAG
Guicao 2hao:TATATTTTTCTGTCCCTTAAAATAGTTTTTTTTTTCTATTACTGTCATTCA TGTGAC
TCATTTTGTTTTTTAG
Jiayu 948:TATATTTTTCTGTCCCTTAAAATAGTTTTTTTTTTCTATTACTGTCATTCAT GTGACTC
ATTTTGTTTTTTAG
Huanjienuo:TATATTTTTCTGTCCCTTAAAATAGTTTTTTTTTTCTATTACTGT CATTCATGTGACT
CATTTTGTTTTTTAG
Yuxian 7hao:TATATTTTTCTGTCCCTTAAAATAGTTTTTTTTTTCTATTACTGTCATTCA TGTGAC
TCATTTTGTTTTTTAG
Fengxinzhan:TATATTTTTCTGTCCCTTAAAATAGTTTTTTTTTTCTATTACTG TCATTCATGTGAC
TCATTTTGTTTTTTAG
Yixiang 1B:TATATTTTTCTGTCCCTTAAAATAGTTTTTTTTTTCTATTACTGTCATTCATG TGACT
CATTTTGTTTTTTAG
Minghui63:TATATTTTTCTGTCCCTTAAAATAGTTTTTTTTTTCTATTACTGTC ATTCATGTGACTC
ATTTTGTTTTTTAG
Zinuo:TATATTTTTCTGTCCCTTAAAATAGTTTTTTTTTTCTATTACTGTCATTC ATGTGACTCATTT
TGTTTTTTAG
Minghui81:TATATTTTTCTGTCCCTTAAAATAGTTTTTTTTTTCTATTACTGTC ATTCATGTGACTC
ATTTTGTTTTTTAG
(2) design primer:
According to the design principle of InDel labelling, the upstream and downstream in described InDel site designs primer, and primer is to base
Sequence is as follows:
F1:5 '-GTTTCGAGATAGTGCTAA-3 '；
R1:5 '-GAATGACAGTAATAGAAA-3 '.
(3) PCR amplification, it is thus achieved that containing the fragment of Pi5 gene specific InDel
Utilize above-mentioned primer to F1 and R1, (use CTAB method to extract plant base with the STb gene of 12 above-mentioned rice varieties
Because group DNA obtains) it is template, carry out standard PCR amplification, amplified production, after 1% agarose gel electrophoresis detection, is connected to
On Pmd18-T Vector, choose PCR method and confirm to check order containing the carrying out inserting purpose fragment that (Sai Mo flies your scientific and technological limited public affairs of generation
Department).
Pcr amplification reaction system is as follows:
2x Reaction Mix:12.5 μ L
Primers F 1 (10 μMs): 1 μ L
Primer R1 (10 μMs): 1 μ L
Golden DNA polymerase:0.4 μ L
DNA profiling (20-50ng/ μ L): 1 μ L
ddH2O: complement to 25 μ L.
PCR Thermal cycling conditions is as follows: 94 DEG C 3 minutes；94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulations；72
DEG C 5 minutes；10 DEG C of preservations.
After PCR reaction terminates, take appropriate enzyme action sample on the agarose gel of 1%, carry out electrophoresis detection, deposition condition
For 90V, 20 minutes, kits.
(4) PCR primer connects, checks order
1) in microcentrifugal tube, following DNA solution is preparedFull dose is 5 μ L.
18-T Vector*1 1μL
Purified pcr product 4 μ L
2) the Solution I of 5 μ L equivalent is added.
3) 16 DEG C are reacted 30 minutes.
4) full dose 10 μ L adds to 100 μ L JM109 competent cells, places 30 minutes in ice.
5) 42 DEG C heating 45 seconds afterPlace 1 minute in ice again.
6) 890 μ L SOC culture medium are added, 37 DEG C of shaken cultivation 60 minutes.
7) cultivate on the L-Agar Plating containing Amp, form single bacterium colony.
8) select white colony, use PCR method to confirm the length scale of Insert Fragment in carrier.
Embodiment 2: resistant gene Pi5 gene specific molecular marker is differentiating the application of Pi5 gene.
PCR primer band according to polymorphism, can be making a distinction containing target gene Pi5.Fig. 1 is to utilize to develop
The labelling JJ801 amplification situation to 12 representative kinds, according to the foundation of this Marker Identification, IRBL5-M (Pi5), middle flower
16, open country, Guangdong accounts for, flower joint extensive 63, osmanthus glutinous, bright is all positive banding pattern towards No. 2 and round-grained rice 7.The result of Fig. 2 shows, Pi5 gene is special
Opposite molecule labelling can distinguish the allele not comprising this site, can distinguish again the allele comprising this site.The most just
It is to say, every rice varieties carrying Pi5, electrophoresis detection band presents with 93bp, and other equipotential base on this site
Because presenting with 95bp, do not comprise this site to present without band.Visible result of the test relatively before labelling accuracy be improved,
Illustrate that resistant gene Pi5 gene specific molecular marker is applied in terms of differentiating Pi5 gene.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify,
All should be the substitute mode of equivalence, within being included in protection scope of the present invention.
SEQUENCE LISTING
<110>Huaiyin Normal College
Jiangsu Province Agriculture Science Institute
<120>resistance gene of rice blast Pi5 specific Function molecular marker and application thereof
<130>
<160> 18
<170> PatentIn version 3.3
<210> 1
<211> 93
<212> DNA
<213>artificial sequence
<400> 1
gtttcgagat agtgctaata taaattggca tgaatagtaa taatatattt ttctgtccct 60
taaaatagtt ttttttttct attactgtca ttc 93
<210> 2
<211> 18
<212> DNA
<213>artificial sequence
<400> 2
gtttcgagat agtgctaa 18
<210> 3
<211> 18
<212> DNA
<213>artificial sequence
<400> 3
gaatgacagt aatagaaa 18
Claims (6)
1. resistance gene of rice blast Pi5 specific Function molecular marker, it is characterised in that named Pi5InDel, nucleic acid sequence
Row are as shown in SEQ ID NO.1.
Resistance gene of rice blast Pi5 specific Function molecular marker the most according to claim 1, it is characterised in that described
The rice blast resistance kind carrying rice blast resistance gene Pi5 be resistant rice cultivars IRBL5-M.
3. the application in differentiating Rice Blast resistant gene of the molecular marker described in claim 1.
Application the most according to claim 3, it is characterised in that comprise the steps of: (1) expands: utilize primers F l and Rl pair
The genome of rice varieties to be detected carries out PCR；(2) detection: molecular size range be 93bp nucleotide fragments, the most to be checked
The rice varieties surveyed carries rice blast resistance gene Pi5；If being not detected by the nucleotide fragments that molecular size range is 93bp,
Rice varieties the most to be detected does not carry rice blast resistance gene Pi5.
5. for expanding the primer pair of molecular marker described in claim 1, it is characterised in that nucleotide sequence is as follows:
SEQ ID NO.2:Fl:5 '-GTTTCGAGATAGTGCTAA-3 '；
SEQ ID NO.3:Rl:5 '-GAATGACAGTAATAGAAA-3 '.
6. the test kit differentiating Rice Blast resistant gene, it is characterised in that containing nucleotide sequence such as SEQ ID
Pi5InDel shown in NO.1, and the primer pair as described in SEQ ID NO.2 and SEQ ID NO.3.
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Cited By (5)
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| CN106701749A (en) * | 2017-02-24 | 2017-05-24 | 淮阴师范学院 | Rice blast resistance gene Piz-t functional specific molecular marker and detection method and application thereof |
| CN107164547A (en) * | 2017-07-19 | 2017-09-15 | 安徽丰大种业股份有限公司 | A kind of molecular labeling, primer and its application with resistance gene of rice blast close linkage |
| CN108998564A (en) * | 2018-09-25 | 2018-12-14 | 湖南杂交水稻研究中心 | InDel molecular marked compound, detection method and the application of blast resistant gene Pid3 |
| CN112501339A (en) * | 2020-12-11 | 2021-03-16 | 华智生物技术有限公司 | SNP molecular marker of rice blast resistance gene Pi5 and application thereof |
| CN113151540A (en) * | 2021-03-15 | 2021-07-23 | 淮阴师范学院 | Specific molecular marker of rice nuclear male sterility gene OsNP1 genotype and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701749A (en) * | 2017-02-24 | 2017-05-24 | 淮阴师范学院 | Rice blast resistance gene Piz-t functional specific molecular marker and detection method and application thereof |
| CN107164547A (en) * | 2017-07-19 | 2017-09-15 | 安徽丰大种业股份有限公司 | A kind of molecular labeling, primer and its application with resistance gene of rice blast close linkage |
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| CN108998564A (en) * | 2018-09-25 | 2018-12-14 | 湖南杂交水稻研究中心 | InDel molecular marked compound, detection method and the application of blast resistant gene Pid3 |
| CN108998564B (en) * | 2018-09-25 | 2021-05-07 | 湖南杂交水稻研究中心 | InDel molecular marker of rice blast resistance gene Pid3, detection method and application |
| CN112501339A (en) * | 2020-12-11 | 2021-03-16 | 华智生物技术有限公司 | SNP molecular marker of rice blast resistance gene Pi5 and application thereof |
| CN112501339B (en) * | 2020-12-11 | 2023-06-27 | 华智生物技术有限公司 | SNP molecular marker of rice blast resistance gene Pi5 and application thereof |
| CN113151540A (en) * | 2021-03-15 | 2021-07-23 | 淮阴师范学院 | Specific molecular marker of rice nuclear male sterility gene OsNP1 genotype and application thereof |
| CN113151540B (en) * | 2021-03-15 | 2023-08-11 | 淮阴师范学院 | Specific molecular marker of rice nuclear male sterile gene OsNP1 genotype and application thereof |
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