CN106811466B - A kind of more slime mould partings of China's wheat root cereal and its method of identification - Google Patents
A kind of more slime mould partings of China's wheat root cereal and its method of identification Download PDFInfo
- Publication number
- CN106811466B CN106811466B CN201611091432.3A CN201611091432A CN106811466B CN 106811466 B CN106811466 B CN 106811466B CN 201611091432 A CN201611091432 A CN 201611091432A CN 106811466 B CN106811466 B CN 106811466B
- Authority
- CN
- China
- Prior art keywords
- cereal
- seq
- slime
- primer
- wheat root
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- 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
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of more slime mould partings of China's wheat root cereal and its methods of identification, the method is to carry out PCR amplification to wheat root genomic DNA using the specific forward primer PSP2 (f) and reverse primer ITS4 (r) of the more slime moulds of cereal, judges wheat root with the presence or absence of the more slime moulds of cereal.Forward primer NS7 (f) and reverse primer ITS.CN (r) and forward primer Pg.F2 (f) and reverse primer Pg.R2 (r) is recycled to determine the parting of the more slime moulds of cereal.
Description
Technical field
The present invention relates to the molecular detection technologies of crop disease virus disease vector, belong to technical field of agriculture science.
Background technique
The more slime moulds of cereal (Polymyxa graminis) are that a kind of low equal eukaryons of the obligatory parasitism of gramineae plant root are raw
Object, itself will not generate apparent harm to the growth of plant, but it is as at least 11 kinds of viral vectors, propagation
Virus can cause a variety of virus diseases on the crops such as wheat, barley, rice.Because of its living body parasitic character, room not can be carried out
Interior in vitro culture, resting spore wall thickness, resistance is strong, can survive in the soil more than ten years, leads to the research to the more slime moulds of cereal
It is very difficult, it makes slow progress.By worldwide a large amount of samplings and identification, the wheat disease that the more slime moulds of cereal are propagated is specified
The malicious Bymovirus that is mainly under the jurisdiction of belongs to and Furovirus category.In the Winter Wheat Area in China, on the more slime mould main propagation wheats of cereal
Wheat yellow mosaic virus (WYMV) and Chinese wheat mosaic virus (CWMV), cause wheat plant tiller reduce, downgrade jaundice
Etc. symptoms, can lead to the underproduction 50-70% when falling ill serious, endanger huge.
There are two kind, the more slime moulds of cereal and the more slime moulds of beet under more slime moulds.The two species initially mainly pass through form
And host distinguishes, the more slime moulds of cereal mainly infect gramineous crop, and the host of the more slime moulds of beet is then chenopod.Due to standing grain
The more slime mould genomic informations of paddy are unknown, and the classification under its kind at present depends on the ITS1 and ITS2 of rDNA (rDNA)
Regional sequence.Ward in 1994 etc. utilize restriction fragment length polymorphism (RFLP) technology, first passage ITS distinguished this two
Kind more slime moulds, while the more slime moulds of cereal are found by sequence analysis there are two kinds of ribotypings.Then, Ward and Adams exist
The third parting of the more slime moulds of cereal is had found on India sorghum.With the announcement of the more slime mould ITS of more and more cereals, Ward
Phyletic evolution point is carried out Deng by the more slime mould ITS sequences of cereal known to African Territories, Asia Japan Area sample and remaining area
Discovery shares the more slime mould partings of 6 kinds of cereals (I type-VI type) at present after analysis.Legreve in 2003 and the research that she works together then show
The parting foundation of the more slime moulds of cereal is other than ITS sequence, and the more slime mould partings of the cereal in different regions source are to host and temperature
There are different requirements, also needs to pay attention in parting.The entire history of life is completed on sorghum from the more slime moulds of the cereal of India
Optimum temperature be 27-30 DEG C, the sprouting of 23-26 DEG C of resting spore and the rate of development in each stage slow down, and 19-22 DEG C is infected
Not significant, 19 DEG C or less nothings infect.And optimum temperature of the more slime moulds of cereal in Temperate Region in China source on barley is 15-18 DEG C,
19-22 DEG C of infection rate is substantially reduced, and 22 DEG C or more are infected not significantly.Therefore, Legreve is mostly glutinous to cereal according to its result of study
When bacterium progress parting in addition to considering ITS sequence, it is also added into host and temperature factor, the more slime moulds of cereal have been divided into five kinds
Type (f.sp.temperate, f.sp.tepida, f.sp.tropicalis, f.sp.subtropicalis,
f.sp.colombiana).Although the two current categorizing systems are not unified, its corresponding relationship is clearly.
Although the communication media of the wheat yellow mosaic in clear China winter wheat area is soil early in the eighties in last century
More slime moulds of cereal in earth, but few to the research of the more slime moulds of cereal, there are which partings for the more slime moulds of China cereal at present not yet
Know, while not knowing whether the Wheat Virus type of its parting and propagation is related.Therefore, the outburst of China's wheat yellow mosaic is specified
The prevention and control that the parting of the more slime moulds of region cereal helps to pass wheat yellow mosaic for soil provide foundation.
Summary of the invention
This research provides a kind of method of Chinese cereal more slime mould parting Rapid identifications and differentiation.According to the more slime moulds of cereal
Ribosomal dna sequence is expanded using the Winter Wheat Area wheat root DNA in known cereal more slime mould primer pairs China, screening
Suitable for the primer of the more slime mould partings of China cereal, while for the anti-of the more slime mould parting design specificity of the distinctive cereal in China
Reaction system and amplification condition to primer I TS.CN, and to PCR make a large amount of optimizations, and final obtain can stablize amplification and identify me
The method of the more slime mould partings of two kinds of cereals of state.
Researcher of the invention detects the region cereal more slime mould rDNA (rDNA) ITS2 first with European Region
Universal primer PSP2 (f) and ITS4 (r) to derived from China's wheat yellow mosaic area wheat root genomic DNA carry out PCR
It expands (PCR product about 250bp), amplified production sequencing simultaneously carries out phylogenetic analysis, preliminary clear China according to the region ITS2
At least there are two kinds of partings in the more slime moulds of Winter Wheat Area cereal, a kind of and reported I type has certain homology, be that China is distinctive
Parting, is named as I-CN type bacterium, and another kind is II type bacterium.For the complete ITS sequence (packet for further obtaining the more slime mould rDNA of cereal
Include ITS1 and ITS2) for distinguishing two kinds of bacterial types, use the general forward primer NS5 of the reported ITS sequence of European Region
(f), NS7 (f), PSP1 (f), ITS5 (f) and reverse primer ITS4 (r) combination are expanded.But wheat is arrived in main amplification
RDNA sequence fails to stablize specifically amplification to the more slime mould ITS sequences of cereal.Therefore, researcher of the invention devises
A large amount of primer finally screens to obtain a reverse primer ITS.CN (r), includes the pact of ITS1 using forward primer NS7 amplification
800bp sequence can be used for detecting the specific primer of I-CN type bacterium.In addition, utilizing II type bacterium primer Pg.F2 (f) and Pg.R2
(r) amplification obtains the special ITS sequence of II type bacterium, and PCR product length is 430bp.The specific present invention uses following technical side
Case:
1. the acquisition of sample
Field morbidity wheat samples are acquired, blade virus is detected by RT-PCR and micro- sem observation root cereal is mostly glutinous
The resting spore situation of bacterium specifies sample root and contains the more slime moulds of cereal.
2. the determination of the more slime mould partings of China cereal
The more slime mould ITS2 sequences of wheat root cereal are expanded first with primer combination PSP2 (f) and ITS4 (r), are sequenced bright
True specific extension increasing sequence constructs chadogram using the more slime mould partings of cereal known on NCBI, specifies the more slime moulds of China cereal
Parting is I-CN type and II type.
3. the specific primer design of parting
According to the sequence characteristic design primer ITS.CN (r) of the distinctive parting I-CN type in China, in conjunction with forward primer NS7
(f) PCR specific amplification I-CN type.Utilize Pg.F2 (f) and Pg.R2 (r) PCR specific amplification II type bacterium.
4. the specificity verification of primer
It extracts the wheat old complaint total DNA for containing the different more slime moulds of parting cereal and verifies primer specific as template PCR amplification
Property, the specific primer for finally clearly detecting the more slime moulds of cereal is PSP2 (f) and ITS4 (r), the detection more slime mould I type bacterium of cereal
Specific primer is Ns7 (f) and ITS.CN (r), the specific primer of the detection more slime mould II type bacterium of cereal be Pg.F2 (f) and
Pg.R2(r)。
5. the optimization of reaction system and amplification condition
To each condition for influencing PCR amplification: including annealing temperature, primer concentration, HiFi archaeal dna polymerase concentration, dNTP
Concentration, extension of time and cycle-index etc. optimize, final to determine the best PCR system for being suitble to this research are as follows: 10 ×
1 μ L, dNTP 2mM of PCR Buffer 1 μ L, 10 μM of 0.25 μ L of upstream primer, 10 μM of 0.25 1.0 μ of μ L, DNA of downstream primer
0.25 6.25 μ L of μ L, ddH2O of L, HiFi DNA polymerase 5U/ μ L, final volume are 10 μ L；Best PCR amplification program
Are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 58 DEG C of annealing 50s, 72 DEG C of extension of time are respectively as follows: PSP2 according to primer difference
(f) extend 20s, Ns7 (f) and ITS.CN (r) with ITS4 (r) and extend 60s, Pg.F2 (f) and Pg.R2 (r) extension 30s, carry out altogether
35 circulations；Last 72 DEG C of extensions 10min.
5. application under study for action
More comprehensive investigation is carried out using the more slime mould parting situations of cereal of the technology to winter wheat area, China, sample comes
Source includes 66 wheat old complaint samples that Shandong, Henan, Jiangsu, Anhui and five, Hubei province soil pass wheat yellow mosaic lesion
Product, the case where specifying the various regions sample more slime moulds of band cereal and its parting.
Using technical solution of the present invention, following technical effect can be obtained:
1. obtaining the specific primer of identification cereal more slime mould I-CN types and II type；
2. obtaining the PCR system of stable detection cereal more slime mould I-CN types and II type bacterium.
Detailed description of the invention
(PSP2 and ITS4 are position view of three pairs of primers of the Fig. 1 for detecting on the more slime mould rDNAs of cereal
The more slime mould specific primers of cereal；NS7 and ITS.CN is the more slime mould I-CN type-special primers of cereal；Pg.F2 and Pg.R2 is standing grain
The more slime mould II type-special primers of paddy；→ indicate forward primer；← indicate reverse primer)
Sample detection effect (the M:Marker of the more slime mould parting specific primers of Fig. 2 cereal；A:PSP2/ITS4 primer sets
Close the more slime moulds of specific amplification cereal；NS7/ITS.CN primer combines the more slime moulds of specific amplification I-CN type cereal；Pg.F2/
Pg.R2 primer combines the more slime moulds of specific amplification II type cereal；The wheat root that the more slime moulds of sample A:I-CN type cereal are infected；Sample
The wheat root that product B:I-CN type and the more slime moulds of II type cereal are infected jointly；Sample C: the wheat root that the no more slime moulds of cereal are infected
Portion；Sample D: blank control (H2O))
Specific embodiment
Come that the present invention is furture elucidated below by the detailed description of specific embodiment, but is not limit of the invention
System, only illustrates.
Embodiment 1: design, synthesis and the PCR Preliminary detection of specific primer
Following forward primer: NS5 is designed with parting specific primer sequence according to the more slime moulds of reported cereal are general,
NS7, PSP1, ITS5, PSP2, Pg.F2；And reverse primer: ITS4, Pg.R2.Preliminary PCR detects lesion wheat root sample
Afterwards, the sequence that band occurs in electrophoresis is tapped and recovered and connects carrier T sequencing.Pairing, sequence and the preliminary PCR of primer
As a result as shown in table 1 below:
1, table is studied primer sequence used and its PCR result
Note: primer entrusts Beijing Qing Kexin industry Bioisystech Co., Ltd to synthesize.
Sequencing result shows that the more slime mould universal primer PSP2 (f) of cereal/ITS4 (r) can be expanded smoothly to the more slime moulds of cereal
RDNA ITS2 sequence, the ITS2 sequencing to a large amount of wheat samples in China Winter Wheat Area (five provinces totally 66 parts of samples, detailed in Example 3)
At least there are two kinds of partings in the interpretation of result tentatively more slime moulds of clear China Winter Wheat Area cereal, a kind of and reported I type has one
Determine homology, be the distinctive parting in China, be named as I-CN type bacterium, another kind is II type bacterium.Pg.F2 (f)/Pg.R2 (r) is same
Can specificity amplification to the more slime mould sequences of cereal (II type).
Meanwhile NS5 (f)/ITS4 (r), NS7 (f)/ITS4 (r), PSP1 (f)/ITS4 (r), ITS5 (f)/ITS4 (r),
Although PSP2 (f)/ITS4 (r) primer combination can be expanded smoothly to correct size strip, sequencing result is wheat rDNA, rather than
The more slime mould rDNA of cereal.Further sequence alignment result shows the Duan Xu in reverse primer ITS4 sequence and wheat rDNA
Column exact matching.Thus while reported ITS4 sequence can be widely used in the more slime mould detections of cereal in Europe, but in China
It is not suitable on wheat, this may be because the crop that Europe is used for the more slime mould detections of cereal at present is mainly that barley, rye etc. make
Object, rather than wheat.
Based on result above, according to the homology of wheat and the more slime mould I-CN type rDNA sequences of cereal, in the difference of the two
One reverse primer ITS.CN of Position Design, utilizing forward primer NS7 amplification includes the about 800bp sequence of cereal more slime mould ITS1
Column, for detecting the specific primer (being shown in Table 1) of I-CN type bacterium.Therefore, it by the screening to the more slime mould primers of cereal, obtains
For universal primer PSP2 (f)/ITS4 (r) of the more slime mould detections of China cereal, the more slime moulds of newfound China I-CN type cereal
Specific primer NS7 (f)/ITS.CN (r) and specific primer Pg.F2 (f)/Pg.R2 (r) of II type bacterium.
Embodiment 2: the PCR system and condition of the more slime moulds of detection cereal and its parting are established
Clip does every part of 30mg of old complaint, is extracted using the DNAsecure Plant Kit kit method of TIANGEN company
The total DNA of wheat old complaint sample is dissolved in 50 μ L ddH2O.
Carry out the PCR reaction of 50 μ L systems, system are as follows: 10 × PCR Buffer, 5 μ L, dNTP 2mM, 5 μ L, upstream primer
10 μM of 1.25 μ L, downstream primer 10 μM of 1.25 μ L, DNA5 μ L, HiFi DNA polymerase 5U/ μ L 0.5 μ L, ddH2O
32 μ L, final volume are 50 μ L；PCR amplification program are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 58 DEG C of annealing 50s, 72 DEG C are prolonged
Stretching the time is respectively as follows: PSP2 (f) and ITS4 (r) according to primer difference and extends 20s, Ns7 (f) and ITS.CN (r) extension 60s,
Pg.F2 (f) and Pg.R2 (r) extends 30s, carries out 35 circulations altogether；Last 72 DEG C of extensions 10min.PCR product is subjected to electrophoresis
It analyzes and observes in the UV lamp, it can be observed that the stripe size that primer PSP2 (f) and ITS4 (r) are amplified is about 250bp,
The stripe size that primer Ns7 (f) and ITS.CN (r) are amplified is about what 800bp, primer Pg.F2 (f) and Pg.R2 (r) were amplified
Stripe size is about 430bp.
Specific fragment is subjected to gel recycling respectively, Cloning Transformation is carried out, transfers to Beijing to hold up the positive colony of acquisition
Ke Xin industry Bioisystech Co., Ltd carries out sequencing analysis.Sequencing result is respectively as follows:
The extension increasing sequence (SEQ ID NO:10) of primer PSP2 (f) and ITS4 (r):
TTGCGCTTTCGAGAGCCCTCGGAAGCATGCCTCTCTGAGTATCGGTTTCTATATCTCTCACATACTCAC
TGGTGGTGTGTGTGGTGGAGAATGGGAGTTGCGCACGTCTCTCTGCATCTCCTGAAGTCAGCGTCGGCCGAAGTCCA
TCGGGTTCTTGGAACGATAATCCGTCATGGCGCCCAAACCATACACAAAAGATCTCAGATGAGGTAAGACTACCCGC
CGAATTTAAGCATATCAATAAGCGGAGGA
The extension increasing sequence (SEQ ID NO:11) of primer Ns7 (f) and ITS.CN (r):
GAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACAATGCTAGGTTCAA
CGAGTCGATTTCGTACTTGGCCGAGAGGCCTGGTTAATCTTCTGAAATCCTAGCGTGCTTGGGCTTGCCTCTTGCAA
CTAGAGGCACCAACGAGGAATTCCTAGTAGACGCAGGTCATCAACCTGCATCGATTACGTCCCTGCCCTTTGTACAC
ACCGCCCGTCGCTTCTACCGATTGGTCGTCCCGGTGAACAATCGGGAGGCCGTGCTCATGGTCAGCAATGGCCGGAG
CGCGGTCGAACTTTTGTAAATTTGGACGACTAGAGGAAGAAGAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGG
AAGGATCATTAGCGTTGATTGGTCTTGGTGCCATGCGAAAAACATGTGGATCGTGGGCTATGTGACCCCTCTGTGGG
GGGTATTTTGTCGCGAACTTGGAGATTTGGCTGATGGATATACATGATAGATAAACAAAATACAACTCTTAGCAATG
GATATCTTGGTTCCCACAACGATGAAGAACGCAGCGAAATGCGATACGTAATGCGAATTGCAGAATTCAGTGAATCA
TCGAATCTTTGAACGCAAGTTGCGCTTTCGAGAGCCCTCGGAAGCATGCCTCTCTGAGTATCGGTTTCTATATCTCT
CACACACTCACTGGTGGTGTGTGTGGTGGAGAATGGGAGTTGCGCACGTCTCTCTGCATCTCCTGAAGTCAGCGTCG
GCCGAAGTCCATCGGGTTCTTGGAACGATAATCCGC
The extension increasing sequence (SEQ ID NO:12) of primer Pg.F2 (f) and Pg.R2 (r):
ATGTGGATCGTCTCTGTTGCTGGATACCGGGATGGAACGCCCTCGTGGTGGTTCTTGTCTTTCACGAAT
TGGATCAAACGGTGGCTAAAGTCGAATTGGTTTCATAACCTACAAACAATATATACAACTCTTAGCAATGGATATCT
TGGTTCCCACAACGATGAAGAACGCAGCGAAATGCGATACGTAATGCGAATTGCAGAATTCAGTGAATCATCGAATC
TTTGAACGCAAGTTGCGCTTTCGAGAGCCCTCGGAAGCATGCCTCTCTGAGTATCGGTTTCCCATCTCACACCCGGT
GTGGAGAATGAGGGTCGGGCGCCTCGGCGCCGGTCCCTTCAAATCAGGCCGGCCTGGTCAAGTCCATCGGATTGCTG
GTACGATAGTCCGTCATGGCGCAAGGAACCACTTGGCAAGATCTCAGATGAGG
Embodiment 3:
Application of the detection method in practical study in the present invention
It is had detected using the technology including from Shandong, Henan, Jiangsu, Anhui, five, Hubei province wheat yellowtop disease
62 wheat old complaint samples in area, the case where specifying the various regions sample more slime moulds of band cereal and its I type and II type bacterium.It can from table
To find out, the more slime moulds of I type cereal are all distributed in this five provinces, II type bacterium is distributed in small part place simultaneously, but simultaneously
Not the case where not finding individualism II type bacterium.
Amino acid sequence table
<110>Zhejiang Academy of Agricultural Science
<120>a kind of more slime mould partings of China's wheat root cereal and its method of identification
<130> NKY001
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> Artificial
<220>
<223> NS5(f)
<400> 1
aacttaaagg aattgacgga ag 22
<210> 2
<211> 20
<212> DNA
<213> Artificial
<220>
<223> ITS4(r)
<400> 2
tcctccgctt attgatatgc 20
<210> 3
<211> 23
<212> DNA
<213> Artificial
<220>
<223> NS7(f)
<400> 3
gaggcaataa caggtctgtg atg 23
<210> 4
<211> 20
<212> DNA
<213> Artificial
<220>
<223> PSP1(f)
<400> 4
tagacgcagg tcatcaacct 20
<210> 5
<211> 22
<212> DNA
<213> Artificial
<220>
<223> ITS5(f)
<400> 5
ggaagtaaaa gtcgtaacaa gg 22
<210> 6
<211> 19
<212> DNA
<213> Artificial
<220>
<223> PSP2(f)
<400> 6
ttgcgctttc gagagccct 19
<210> 7
<211> 19
<212> DNA
<213> Artificial
<220>
<223> Pg.F2(f)
<400> 7
ttgcgctttc gagagccct 19
<210> 8
<211> 23
<212> DNA
<213> Artificial
<220>
<223> Pg.R2(r)
<400> 8
cctcatctga gatcttgcca agt 23
<210> 9
<211> 20
<212> DNA
<213> Artificial
<220>
<223> ITS.CN(r)
<400> 9
gcggattatc gttccaagaa 20
<210> 10
<211> 252
<212> DNA
<213>the more slime moulds of cereal (Polymyxa graminis)
<400> 10
ttgcgctttc gagagccctc ggaagcatgc ctctctgagt atcggtttct atatctctca 60
catactcact ggtggtgtgt gtggtggaga atgggagttg cgcacgtctc tctgcatctc 120
ctgaagtcag cgtcggccga agtccatcgg gttcttggaa cgataatccg tcatggcgcc 180
caaaccatac acaaaagatc tcagatgagg taagactacc cgccgaattt aagcatatca 240
ataagcggag ga 252
<210> 11
<211> 798
<212> DNA
<213>the more slime moulds of cereal (Polymyxa graminis)
<400> 11
gaggcaataa caggtctgtg atgcccttag atgttctggg ccgcacgcgc gctacaatgc 60
taggttcaac gagtcgattt cgtacttggc cgagaggcct ggttaatctt ctgaaatcct 120
agcgtgcttg ggcttgcctc ttgcaactag aggcaccaac gaggaattcc tagtagacgc 180
aggtcatcaa cctgcatcga ttacgtccct gccctttgta cacaccgccc gtcgcttcta 240
ccgattggtc gtcccggtga acaatcggga ggccgtgctc atggtcagca atggccggag 300
cgcggtcgaa cttttgtaaa tttggacgac tagaggaaga agaagtcgta acaaggtttc 360
cgtaggtgaa cctgcggaag gatcattagc gttgattggt cttggtgcca tgcgaaaaac 420
atgtggatcg tgggctatgt gacccctctg tggggggtat tttgtcgcga acttggagat 480
ttggctgatg gatatacatg atagataaac aaaatacaac tcttagcaat ggatatcttg 540
gttcccacaa cgatgaagaa cgcagcgaaa tgcgatacgt aatgcgaatt gcagaattca 600
gtgaatcatc gaatctttga acgcaagttg cgctttcgag agccctcgga agcatgcctc 660
tctgagtatc ggtttctata tctctcacac actcactggt ggtgtgtgtg gtggagaatg 720
ggagttgcgc acgtctctct gcatctcctg aagtcagcgt cggccgaagt ccatcgggtt 780
cttggaacga taatccgc 798
<210> 12
<211> 430
<212> DNA
<213>the more slime moulds of cereal (Polymyxa graminis)
<400> 12
atgtggatcg tctctgttgc tggataccgg gatggaacgc cctcgtggtg gttcttgtct 60
ttcacgaatt ggatcaaacg gtggctaaag tcgaattggt ttcataacct acaaacaata 120
tatacaactc ttagcaatgg atatcttggt tcccacaacg atgaagaacg cagcgaaatg 180
cgatacgtaa tgcgaattgc agaattcagt gaatcatcga atctttgaac gcaagttgcg 240
ctttcgagag ccctcggaag catgcctctc tgagtatcgg tttcccatct cacacccggt 300
gtggagaatg agggtcgggc gcctcggcgc cggtcccttc aaatcaggcc ggcctggtca 360
agtccatcgg attgctggta cgatagtccg tcatggcgca aggaaccact tggcaagatc 420
tcagatgagg 430
Claims (8)
1. a kind of more slime mould partings of China's wheat root cereal, which is characterized in that the nucleotides sequence indicated for SEQ ID NO:11
Column.
2. a pair of of specific primer of the more slime mould partings of cereal described in claim 1, which is characterized in that forward primer SEQ
ID NO:3 and reverse primer are SEQ ID NO:9.
3. the detection method of a kind of pair of more slime moulds of wheat root cereal, which is characterized in that utilize forward primer SEQ ID NO:3
With the parting of the more slime moulds of the reverse primer clear cereal of SEQ ID NO:9.
4. the detection method of a kind of pair of more slime moulds of wheat root cereal, which is characterized in that the specificity using the more slime moulds of cereal is drawn
Object SEQ ID NO:6 and SEQ ID NO:2 carries out PCR amplification to wheat root genomic DNA, judges that wheat root whether there is
The more slime moulds of cereal recycle point of forward primer SEQ ID NO:3 and the more slime moulds of the reverse primer clear cereal of SEQ ID NO:9
Type.
5. the identification method of a kind of pair of more slime moulds of wheat root cereal, which is characterized in that the specificity using the more slime moulds of cereal is drawn
Object SEQ ID NO:6 and SEQ ID NO:2 carries out PCR amplification to wheat root genomic DNA, judges that wheat root whether there is
The more slime moulds of cereal recycle forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:9 and forward primer SEQ ID
The parting of NO:7 and the more slime moulds of the reverse primer clear cereal of SEQ ID NO:8.
6. identification method according to claim 5, which is characterized in that PCR reaction system are as follows: 10 × PCR Buffer, 1 μ
1 μ L of L, dNTP 2mM, 10 μM of 0.25 μ L of upstream primer, 1.0 μ L, HiFi DNA of downstream primer 10 μM of 0.25 μ L, DNA
Polymerase 5U/ μ L 0.25 μ L, ddH26.25 μ L of O, final volume are 10 μ L；PCR amplification program are as follows: 94 DEG C of initial denaturations 3
min；94 DEG C of denaturation 30s, 58 DEG C of annealing 50s, 72 DEG C of extension of time are respectively as follows: SEQ ID NO:6 and SEQ according to primer difference
ID NO:2 extends 20s, and SEQ ID NO:3 and SEQ ID NO:9 extends 60s, and SEQ ID NO:7 and SEQ ID NO:8 extends
30s carries out 35 circulations altogether；10 min of last 72 DEG C of extensions.
7. identification method according to claim 5, which is characterized in that the judgement wheat root is more with the presence or absence of cereal
Slime mould refers to: the amplified production according to primer SEQ ID NO:6 and SEQ ID NO:2 be size whether the DNA for being 250bp or so
Band, thus judges whether wheat root contains the more slime moulds of cereal；According to the expansion of primer SEQ ID NO:3 and SEQ ID NO:9
Volume increase object is the DNA band whether size is 800bp or so, and it is mostly glutinous thus to judge whether wheat root contains I-CN type cereal
Bacterium.
8. identification method according to claim 5, which is characterized in that the judgement wheat root is more with the presence or absence of cereal
Slime mould refers to: the amplified production according to primer SEQ ID NO:7 and SEQ ID NO:8 be size whether the DNA for being 430bp or so
Band, thus judges whether wheat root contains the more slime moulds of II type cereal.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611091432.3A CN106811466B (en) | 2016-12-01 | 2016-12-01 | A kind of more slime mould partings of China's wheat root cereal and its method of identification |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611091432.3A CN106811466B (en) | 2016-12-01 | 2016-12-01 | A kind of more slime mould partings of China's wheat root cereal and its method of identification |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106811466A CN106811466A (en) | 2017-06-09 |
| CN106811466B true CN106811466B (en) | 2019-10-11 |
Family
ID=59106138
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611091432.3A Expired - Fee Related CN106811466B (en) | 2016-12-01 | 2016-12-01 | A kind of more slime mould partings of China's wheat root cereal and its method of identification |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106811466B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DD268256A1 (en) * | 1987-12-04 | 1989-05-24 | Adl D Ddr Inst F Phytopatholog | METHOD FOR DETECTING PILZ-TRANSFERABLE VIRUSES FROM GROUND SAMPLES |
| CN102424835A (en) * | 2011-11-10 | 2012-04-25 | 江苏省农业科学院 | Method for detecting Polymyxa graminis in soil |
-
2016
- 2016-12-01 CN CN201611091432.3A patent/CN106811466B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DD268256A1 (en) * | 1987-12-04 | 1989-05-24 | Adl D Ddr Inst F Phytopatholog | METHOD FOR DETECTING PILZ-TRANSFERABLE VIRUSES FROM GROUND SAMPLES |
| CN102424835A (en) * | 2011-11-10 | 2012-04-25 | 江苏省农业科学院 | Method for detecting Polymyxa graminis in soil |
Non-Patent Citations (3)
| Title |
|---|
| Analysis of ribosomal DNA sequences of Polymyxa species;WARD等;《Mycol.Res.》;19981231;第102卷(第8期);第965-974页 * |
| Ribotypes of Polymyxa graminis in Wheat Samples Infected with Soilborne Wheat Viruses in China;Xu Y等;《Plant Dis》;20180313;第102卷(第5期);第948-954页 * |
| 大麦黄花叶病及其真菌介体禾谷多粘菌的研究进展;陈剑平;《中国病毒学》;19920331;第7卷(第1期);第1-10页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106811466A (en) | 2017-06-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105112504B (en) | A kind of molecular marker identification method of the high nitrogen use efficiency gene of rice | |
| CN102162011B (en) | Molecule marking method of rice blast-resisting gene | |
| CN107937599B (en) | Molecular marker KB2 for identifying clubroot resistance of Chinese cabbages, primers and application | |
| CN103305510A (en) | Rice blast resistance gene Pi9 gene specificity molecular marker Pi9SNP as well as preparation and application thereof | |
| CN103497996A (en) | Molecular marker InDe1587 for detecting rice blast resistant gene Pigm(t) of rice #4 | |
| CN104877996A (en) | Haynaldia villosa's 6VS chromosome specific molecular marker 6VS-BH1 and application thereof | |
| CN106555001B (en) | A kind of molecular labeling of rice blast resistant gene and its application | |
| CN104131012A (en) | Molecular marker for identifying soybean nucleus male sterility line and identification method thereof | |
| CN104263813A (en) | Sequences of primer for identifying fusarium solani and/or fusarium oxysporum, kit and method thereof | |
| CN108004346B (en) | Wheat gene Yr10 molecular marker and application thereof in screening wheat with wheat stripe rust resistance | |
| CN110512025A (en) | A kind of molecular labeling and its application with powdery mildew resistance gene in wheat PmJM23 close linkage | |
| CN102796825B (en) | Specificity polymerase chain reaction (PCR) method for detecting heterodera elachista ohshima | |
| CN103866038B (en) | For detecting tobacco to the N gene-specific primer of TMV resistance to, detection method and test kit | |
| CN106811466B (en) | A kind of more slime mould partings of China's wheat root cereal and its method of identification | |
| CN104328205B (en) | The foundation of downy mildew of millet bacterium LAMP method for quick | |
| CN104120124B (en) | SCAR marker for specifically detecting wheat stripe rust and detection method | |
| CN112176080B (en) | Nested PCR primer group, kit and detection method for specifically detecting purple sisal leaf roll disease phytoplasma | |
| CN104059909A (en) | Globodera rostochiensis SCAR marker as well as LAMP fast detection method and application of method | |
| CN114032334A (en) | Primer group and kit for detecting quinoa phomopsis and detection method thereof | |
| CN103409414B (en) | Sugarcane genome endogenous reference gene acetolactate synthase gene PCR (polymerase chain reaction) primer sequences and amplification method | |
| CN107058559B (en) | Molecular detection method and kit for plant pathogenic fungi | |
| KR101149437B1 (en) | Genetic markers for Xanthomonas oryzae pv.oryzae, primers for the markers, and method for detecting and discriminating Xanthomonas oryzae pv.oryzae | |
| CN104404155B (en) | Melon variety breathes out the method for quick of emerald green seed purity | |
| CN103740812A (en) | Method for identification of Avena species A genome and C genome | |
| CN113736796B (en) | Drought-resistant gene, SNP (single nucleotide polymorphism) of shinyleaf yellowhorn and application of drought-resistant gene and SNP |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20191011 Termination date: 20201201 |