CN108624711A - Primer and application of a pair for identifying peaceful wheat No. 9 and its derived varieties scab resistance - Google Patents
Primer and application of a pair for identifying peaceful wheat No. 9 and its derived varieties scab resistance Download PDFInfo
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- CN108624711A CN108624711A CN201810672028.8A CN201810672028A CN108624711A CN 108624711 A CN108624711 A CN 108624711A CN 201810672028 A CN201810672028 A CN 201810672028A CN 108624711 A CN108624711 A CN 108624711A
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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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- 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
- C12Q2600/13—Plant traits
Abstract
The present invention relates to a pair of primer and applications for identifying peaceful wheat No. 9 and its derived varieties scab resistance.The primer nucleotide sequences are as shown in SEQ ID NO.1 and SEQ ID NO.2；PCR amplification is carried out to Wheat volatiles DNA using the primer pair, product is after agarose gel electrophoresis, if the band containing 800 bp in amplified production, then illustrate that the wheat breed to be measured contains the identical scab resistance gene of peaceful wheat No. 9, with scab resistance, conversely, being then susceptible wheat；Wheat breed scab resistance can quickly and be intuitively obtained using the primer as a result, being suitable for extensive breeding screening.
Description
Technical field
It is especially a pair of anti-for screening peaceful wheat No. 9 and its derived varieties head blight this application involves field of wheat breeding
The primer sequence in source and its application.
Background technology
Wheat scab（Fusarium head blight, FHB）It is caused by being main pathogenic bacteria by Fusarium graminearum
A kind of great fungal disease of world wide widespread, and endanger one of the Major Diseases of China's Wheat indurstry development.I
State is one of global maximum country of wheat scab injured area, and traditional diseased region is middle and lower reach of Yangtze River Winter Wheat Area and northeast
Spring wheat area.In recent years, due to the change of climate warming and cropping system, wheat scab morbidity range in China's becomes in constantly expansion
Gesture, at present Chang Faqu expanded to the Yellow River and Huai He River south area of wheat, the Yellow River and Huai He River is northern, also apparent aggravate occurs for southwest, northwest area of wheat disease.
Wheat scab can cause yield to decline, and the general generation time can cause the underproduction of 10%-30%, be damaged when being very popular
Lose bigger, or even total crop failure.Head blight not only causes wheat yield to reduce, and can also influence the quality of wheat, more seriously cause
The secondary metabolite that germ generates after infecting threatens human and livestock health.Therefore, prevention and control wheat scab in active and effective ground is to grain
Food safety and food security are all of great significance.
It is maternal, Japan's wheat breed " west wind " for male parent that peaceful wheat No. 9, which is Jiangsu Province Agriculture Science Institute to raise wheat No. 6,
Hybridization, is formed using bulk method of breeding selection and breeding, because have high yield, stable yields and extensive adaptability, the special quality of high-quality weak muscle and
The features such as anti-wheat yellow mosaic, head blight, it has also become the main breed in Jiangsu Province of China.Peaceful wheat No. 9 is also China's wheat
The important parent of breeding.It is the new variety of wheat that parent has been bred as that a batch is suitable for the plantation of the middle and lower reach of Yangtze River area of wheat with the kind, such as
Peaceful wheat 13, peaceful wheat 18, gives birth to and selects No. 4, gives birth to and select No. 6, raise wheat 18, raise spoke wheat No. 4, town wheat No. 5, town peaceful wheat 14
Wheat No. 8 etc..Peaceful wheat No. 9 has breeding high-yield, high-quality, anti-disease wheat new varieties important as backbone parent of new generation
Value.By selecting specific primer and being applied to the anti gibberellic disease point with peaceful wheat No. 9 and derived varieties for parent
In sub- marker-assisted breeding, the validity of the early generation scab resistance selection of wheat hybridizing breeding is can effectively improve, currently, needle
There is not been reported for the primer detected to peaceful wheat No. 9 and derived varieties scab resistance.
Invention content
In view of the above-mentioned problems, for scab resistance in Rapid identification 9 and its derived varieties, the present invention provides a kind of use
In primer sequence and its application of screening peaceful wheat No. 9 and its anti-source of derived varieties head blight.
The object of the present invention is achieved like this：Anti gibberellic disease base of a pair for identifying peaceful wheat No. 9 and its derived varieties
The primer pair of cause, upstream primer nucleotide sequences are as shown in SEQ ID NO.1, downstream primer nucleotide sequence such as SEQ ID
Shown in NO.2.
It is respectively shown in SEQ ID NO.1 and SEQ ID NO.2 that the present invention additionally provides above-mentioned nucleotide sequence simultaneously
Application of the primer pair in identification identification 9 and its derived varieties scab resistance, i.e., using Wheat volatiles DNA to be measured as template
Pcr amplification reaction is carried out, then amplified production is separated by electrophoresis in Ago-Gel detection, if containing 800 bp in amplified production
The band of left and right size, then wheat to be measured have No. 9 scab resistances of peaceful wheat, otherwise wheat to be measured is without scab resistance.
In the application of above-mentioned primer sequence：
The reaction system of PCR amplification（25 μL）For：The Wheat volatiles DNA to be measured of 20-100ng, the Taq enzyme 0.25 of 5 U/ μ L
μ L, dNTP 0.5 the μ L, 10 × PCR of SEQ ID NO.1 and SEQ ID the NO.2 each 1 μ L, 10 mmol/L of 10 μm of ol/L
2.5 μ L of Mg2+ Plus buffer solutions, surplus is sterile distilled water；
The reaction condition of PCR amplification is：94 DEG C of pre-degeneration 5 min, subsequent 94 DEG C of denaturation 30 sec, anneal 30 under the conditions of 58 DEG C
Sec, 72 DEG C of 40 sec of extension, carries out 35 cycles；5 min of last 72 DEG C of extensions;It is preserved at 4 DEG C.
In the application of above-mentioned primer sequence：
Amplified production is separated by electrophoresis in Ago-Gel detection refers to：By amplified production mass fraction be 0.8%-2%
Ago-Gel（EB containing final concentration of 0.5 μ g/mL）Middle electrophoretic separation, 130 V of voltage, 20 min of electrophoresis time judge
In amplified production whether the band containing 800 bp.
In the application, No. 9 derived varieties of peaceful wheat refer to：It is preferably peaceful wheat 13, peaceful with the kinds that peaceful wheat No. 9 is bred as parent
Wheat 14, peaceful wheat 18 give birth to and select No. 4, give birth to and select No. 6, town wheat No. 5, town wheat No. 8, raise wheat 18 and raise spoke wheat No. 4.
In the application, wheat Evaluation standard of resistance referring to《Wheat breed scab resistance identifies regulation》.
The advantage of the invention is that：PCR expansions are carried out to Wheat volatiles DNA by SEQ ID NO.1 and SEQ ID NO.2
Increase, to the detection that amplified production is marked by agarose electrophoresis, sequenator or native gel electrophoresis need not be utilized
It is detected, it is easy to operate；Bring judgement wheat lines whether red containing peaceful wheat No. 9 so that 800 bp size bars can be amplified
Mildew resistant gene very intuitively need not be by being sequenced or comparing the size of band.
Description of the drawings
Fig. 1：Utilize agarose electrophoresis detection primer SEQ ID NO.1 and SEQ ID NO.2 sense kinds anti-to head blight
Amplification.
M：Molecular weight standard DL2000；1-8 swimming lanes are followed successively by the peaceful wheat of wheat breed No. 9, peaceful wheat No. 13, peaceful wheat No. 14, life
It selects No. 6, raise spoke wheat No. 4, peace agriculture 8455, Clark, A Fu.
Specific implementation mode
Wheat breed, pathogen involved in embodiment（Fusarium graminearum FG0609）It is all from Jiangsu Province Agriculture Science Institute
The wheat research department preservation of cereal crops research institute；
DNA extraction kit is purchased from Tiangeng biochemical technology Co., Ltd, model DP305.
Embodiment 1 is related to specific primer
The present embodiment utilizes peaceful wheat No. 9 and Yangmai No.158 filial generation recombinant inbred lines（RIL）Group carries out scab resistance
QTL is positioned.Recombinant inbred lines material is in the continuous two Growing seasons plantation of 2015-2016,2016-2017 in institute of academy of agricultural sciences of Jiangsu Province
Interior proving ground, and reference《Wheat breed scab resistance identifies regulation》Scab resistance has been carried out to recombinant inbred lines material
Identification.Utilize plant genome DNA extracts kit（Tiangeng DP305）DNA is extracted, 90 k genetic chips of Illumina are utilized
Genome scanning is carried out to test material（Specifying information referring to：Wang etc., 2014, Characterization of
polyploid wheat genomic diversity using a high‐density 90 000 single
nucleotide polymorphism array）, soft using the IciMapping 4.1 to be mapped based on compound mixed linear model
Part carries out QTL positioning analysis, finally found that No. 9 anti gibberellic disease main effect QTLs of peaceful wheat are located at BS00098868_51 and Tdurum_
Between contig80344_144 molecular labelings.Upstream and downstream primer difference is further designed for above-mentioned molecular labeling section site
As shown in SEQ ID NO.1 and shown in SEQ ID NO.2, and the Nanjing bio tech ltd Qing Ke is entrusted to synthesize：
Sense primer SEQ ID NO.1：
5’- ATATGGCACACGCTACATTG -3’；
Downstream primer SEQ ID NO.2：
5’- TTGTTGTGGTGGTCATGTTT -3’。
Specific amplification of 2 primer of embodiment in peaceful wheat No. 9 and derived varieties
Wheat breed to be measured：Peaceful wheat No. 9, peaceful wheat No. 13, peaceful wheat No. 14, it is raw select No. 6, raise spoke wheat No. 4, peace agriculture 8455,
Clark, A Fu.
Detection method：
Respectively using SEQ ID NO.1 and SEQ ID NO.2 as upstream and downstream primer, with Wheat volatiles DNA to be measured（The extraction sides DNA
Method is referring to DNA extraction kit specification）Carry out PCR amplification is carried out for template：
PCR reaction systems are：Taq enzyme 0.25 the μ L, the SEQ ID NO.1 of 10 μm of ol/L of the DNA of 20 ng, 5 U/ μ L and
The 2.5 μ L of Mg2+ Plus buffer solutions of dNTP 0.5 the μ L, 10 × PCR of SEQ ID NO.2 each 1 μ L, 10 mmol/L, then
Reaction system is supplemented to 25 μ L with sterile distilled water；
Response procedures：By reaction system in 94 DEG C of pre-degeneration 5 min, subsequent 94 DEG C of denaturation 30 sec anneal 30 under the conditions of 58 DEG C
Sec, 72 DEG C of 40 sec of extension, carries out 35 cycles；5 min of last 72 DEG C of extensions;It is preserved at 4 DEG C.
By amplified production in 1.5% Ago-Gel（EB containing final concentration of 0.5 μ g/mL）Electrophoretic separation obtains
Testing result such as Fig. 1.
In Fig. 1 amplified productions containing 800 bp size strips have peaceful wheat No. 9, peaceful wheat No. 13, peaceful wheat No. 14, it is raw select No. 6,
Raise spoke wheat No. 4, it was demonstrated that it all contains the scab resistance gene of peaceful wheat No. 9, is disease-resistant variety；And pacify agriculture 8455, Clark and
A Fu does not amplify band, it was demonstrated that it does not contain the scab resistance gene, is susceptible variety.
3 field experiment of embodiment
It is further verified using traditional field resistance identification method, in each wheat breed in blooming stage, is chosen in tassel
One small ear on top is inoculated with 10 μ L and contains 1 × 105Gibberella spore liquid（FG0609）, bagging moisturizing 3 days, each kind
20 wheat heads are inoculated with, the sick small ear rate of each wheat breed is investigated within 21 days after inoculation.
Disease Resistance Identification the result shows that, the sick small ear rate of above-mentioned disease-resistant variety is respectively 20.1%（Peaceful wheat No. 9）、19.9%
（Peaceful wheat No. 13）、33.1%（Peaceful wheat No. 14）、19.0%（Life selects No. 6）With 23.6%（Raise spoke wheat No. 4）, it was demonstrated that its certain moderate resistance
Head blight；And the sick small ear rate of above-mentioned susceptible variety is respectively 89.8%（Pacify agriculture 8455）、76.5%（Clark）With 71.5%（Ah
Husband）, it was demonstrated that its not anti gibberellic disease really, is susceptible variety.
It can be obtained by above-mentioned experimental result：By SEQ ID NO.1 and SEQ ID NO.2 to Wheat volatiles DNA into
Can row PCR amplification, can be directly by agarose electrophoresis, to amplify whether 800 bp size bars bring judgement wheat lines
Containing No. 9 scab resistance genes of peaceful wheat, detection method is easy to operate, and testing result is very intuitive, detection result significant effective.
Presently preferred embodiments of the present invention is illustrated above, but the present invention is not limited to the embodiment,
Those skilled in the art can also be made under the premise of without prejudice to spirit of that invention various equivalent type modifications or
It replaces, as long as these equivalent modifications and replacement also belong to protection scope of the present invention within the spirit that claim limits.
Sequence table
<110>Jiangsu Province Agriculture Science Institute
<120>Primer and application of a pair for identifying peaceful wheat No. 9 and its derived varieties scab resistance
<141> 2018-06-26
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
atatggcaca cgctacattg 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ttgttgtggt ggtcatgttt 20
Claims (4)
1. primer of a pair for identifying peaceful wheat No. 9 and its derived varieties scab resistance, nucleotide sequence is respectively such as SEQ
Shown in ID NO.1 and SEQ ID NO.2.
2. application of the primer as described in claim 1 in identifying peaceful wheat No. 9 and its derived varieties scab resistance.
3. application as claimed in claim 2, which is characterized in that be as follows：
Respectively using SEQ ID NO.1 and SEQ ID NO.2 as primer, PCR expansions are carried out by template of Wheat volatiles DNA to be measured
Increase, and to amplified production into row agarose gel electrophoresis, if
Band containing 800 bp sizes in amplified production then judges that wheat to be measured is containing No. 9 anti gibberellic disease genes of peaceful wheat
Otherwise wheat is susceptible wheat.
4. application according to claim 3, which is characterized in that the PCT amplification systems are as follows：
Taq enzyme 0.25 the μ L, the SEQ ID NO.1 of 10 μm of ol/L of the Wheat volatiles DNA to be measured of 20-100ng, 5 U/ μ L and
The Mg of dNTP 0.5 the μ L, 10 × PCR of SEQ ID NO.2 each 1 μ L, 10 mmol/L2+2.5 μ L of Plus buffer solutions, with nothing
Bacterium distilled water complements to 25 μ L；
The reaction condition of PCR amplification is：94 DEG C of pre-degeneration 5 min, subsequent 94 DEG C of denaturation 30 sec, anneal 30 under the conditions of 58 DEG C
Sec, 72 DEG C of 40 sec of extension, carries out 35 cycles；5 min of last 72 DEG C of extensions;It is preserved at 4 DEG C.
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| CN201810672028.8A CN108624711A (en) | 2018-06-26 | 2018-06-26 | Primer and application of a pair for identifying peaceful wheat No. 9 and its derived varieties scab resistance |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810672028.8A CN108624711A (en) | 2018-06-26 | 2018-06-26 | Primer and application of a pair for identifying peaceful wheat No. 9 and its derived varieties scab resistance |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892307A (en) * | 2010-05-12 | 2010-11-24 | 江苏省农业科学院 | SSCP marker closely linked with major wheat scab resistance QTL and application thereof |
| WO2014152383A1 (en) * | 2013-03-15 | 2014-09-25 | Ohio State Innovation Foundation | Methods for using cryptococcus flavescens strains for biological control of fusarium head blight |
| US9506081B1 (en) * | 2013-01-31 | 2016-11-29 | The United States Of America, As Represented By The Secretary Of Agriculture | Transgene construct to improve Fusarium head blight resistance in wheat and barley |
| CN107338310A (en) * | 2017-07-31 | 2017-11-10 | 中国农业科学院作物科学研究所 | A kind of mark and application method for detecting wheat anti gibberellic disease gene PFT |
| CN107988411A (en) * | 2017-12-21 | 2018-05-04 | 河北省农林科学院粮油作物研究所 | The KASP molecular labelings of wheat scab resistance main effect QTL and its application |
-
2018
- 2018-06-26 CN CN201810672028.8A patent/CN108624711A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892307A (en) * | 2010-05-12 | 2010-11-24 | 江苏省农业科学院 | SSCP marker closely linked with major wheat scab resistance QTL and application thereof |
| US9506081B1 (en) * | 2013-01-31 | 2016-11-29 | The United States Of America, As Represented By The Secretary Of Agriculture | Transgene construct to improve Fusarium head blight resistance in wheat and barley |
| WO2014152383A1 (en) * | 2013-03-15 | 2014-09-25 | Ohio State Innovation Foundation | Methods for using cryptococcus flavescens strains for biological control of fusarium head blight |
| CN107338310A (en) * | 2017-07-31 | 2017-11-10 | 中国农业科学院作物科学研究所 | A kind of mark and application method for detecting wheat anti gibberellic disease gene PFT |
| CN107988411A (en) * | 2017-12-21 | 2018-05-04 | 河北省农林科学院粮油作物研究所 | The KASP molecular labelings of wheat scab resistance main effect QTL and its application |
Non-Patent Citations (3)
| Title |
|---|
| ANGELICA GIANCASPRO等: "Mapping QTLs for Fusarium Head Blight Resistance in an Interspecific Wheat Population", 《FRONT PLANT SCI》 * |
| RAWAT N.等: "Triticum aestivum cultivar Sumai 3 Fhb1 region genomic sequence", 《GENBANK》 * |
| 朱展望等: "中国小麦品种抗赤霉病基因Fhb1的鉴定与溯源", 《作物学报》 * |
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