CN107164547A - A kind of molecular labeling, primer and its application with resistance gene of rice blast close linkage - Google Patents
A kind of molecular labeling, primer and its application with resistance gene of rice blast close linkage Download PDFInfo
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Abstract
The invention discloses a kind of molecular labeling of and resistance gene of rice blast close linkage, expand the primer pair of the molecular labeling and the method for detection Rice Resistance To Rice Blast.The nucleotide sequence of the molecular labeling such as SEQ ID NO：1 shown or SEQ ID NO：Shown in 2.Molecular labeling of the present invention is located at resistant gene Pigm gene internal, and with resistance gene of rice blast close linkage, compared with non-genomic inner marker, molecular labeling of the present invention is not in separation, can more effectively improve breeding efficiency.
Description
Technical field
The present invention relates to biological technical field, a kind of and resistance gene of rice blast close linkage is related in particular to
Molecular labeling, primer and its application.
Background technology
The shortcomings of there is low efficiency of selection and long breeding cycle in the conventional breeding methods based on Phenotypic Selection, in the urgent need to note
Enter molecular approach means, it is auxiliary in expeditiously genotype M8003 line, it could quickly and efficiently cultivate excellent paddy rice
New varieties.With developing rapidly for molecular biology and genomics, molecular marking technique is more widely applied.PCR-based
Molecular labeling such as microsatellite or SSR (simple sequence repeat) etc. have that polymorphic rate is high, stablize relatively, detection side
The features such as method is easy to be quick and easily operated and be widely used.
Show recessive interference etc. with character because molecular marker assisted selection is not easily affected by environmental factors, can be from molecular water
Calm down to selection target character gene, while it is also possible to chain between breaking unfavorable gene and expeditiously polymerize multiple excellent
Well-founded is because in one.Usually said molecular labeling had both included the linked marker of target gene or including target gene itself function
Mark.Molecular labeling auxiliary multiple gene polymerization breeding technique has become paddy rice, a kind of development of seeding corn and other crops breeding research
Trend, with the technology can by gene pyramidings such as high-quality, many anti-and high yields into a small number of key varieties, key is
The practical molecular labeling with objective trait gene or main effect QTL compact linkage can be obtained.
Rice blast is one of important disease of paddy rice, can cause the significantly underproduction, the underproduction 40%~50% when serious, or even
Grain is without receipts.Each rice region in the world uniformly occurs.This disease has generation in various regions, wherein occured as with leaf portion, section portion it is many, can after generation
The different degrees of underproduction is caused, especially panicle blast or section pest occurs early and weighed, and can cause dead ears so that total crop failure.So Study On Rice stem
Maize ear rot cause of disease and occurrence regularity, cultivate disease-resistant variety, reduce Rice Yield Loss Caused significant.
Preventing and treating rice blast mainly has two methods, and one is to use chemical pesticide, unsustainable because cost is high, pollution environment；
Two be to excavate new Resistant germplasm and seed selection broad-spectrum disease resistance new varieties.It is introduced that, 25 blast resistant genes quilts gram are had at present
Grand and Function Identification, but the anti-spectrum of the overwhelming majority is narrow, easily loses disease resistance；A variety of disease-resistant genes are incorporated into a kind, no
Only difficulty large period is long, also results in crop yield and quality reduction.
Anti- pest germplasm screens since 2002, extensively in team of He Zu Chinese groups, from the breeding material originating from China's farm variety
The anti-pest novel site Pigm of a wide spectrum is identified in material.Then they used for 10 years again, this new position of system analysis
The functional mechanism of point.Research finds there is the albumen PigmR and PigmS of 2 functions in Pigm.PigmR is to all detections
Pyricularia grisea Race all has broad spectrum resistance, but rice paddy seed can be made to diminish simultaneously, yield reduction；PigmS can suppress
PigmR disease-resistant function, can but improve rice yield.PigmR and PigmS this to " albumen brother " close linkage in chromosome
An away minor segment in, it is impossible to separate, therefore the kind of seed selection just existing broad spectrum resistance and does not influence final yield
(Epigenetic regulation of antagonistic receptors confers rice blast
resistance with yield balance[J].2017.355(6328):962)。
The content of the invention
The technical problems to be solved by the invention are to provide a kind of and resistance gene of rice blast close linkage molecule
Mark, the method for expanding the primer pair of the molecular labeling and detecting Rice Resistance To Rice Blast.
In order to solve the above-mentioned technical problem, the present invention is adopted the following technical scheme that：A kind of and resistance gene of rice blast
The molecular labeling of close linkage, the nucleotide sequence such as SEQ ID NO of the molecular labeling：1 shown or SEQ ID NO：2 institutes
Show.
A kind of primer pair for expanding above-mentioned molecular labeling, including primer 1 and primer 2, the nucleotide sequence of the primer 1 is such as
SEQ ID NO：Shown in 3, the nucleotide sequence such as SEQ ID NO of the primer 2：Shown in 4.
A kind of method for detecting Rice Resistance To Rice Blast, comprises the following steps：
(1) PCR is expanded：Using the genomic DNA that is extracted from paddy rice to be measured as DNA cloning template, with SEQ ID NO：3
Shown nucleotide sequence and SEQ ID NO：Nucleotide sequence composition shown in 4 is amplimer to entering performing PCR amplification；
(2) identification of amplified production：If gained pcr amplification product is 272bp base fragments, paddy rice to be measured is anti-rice blast
Sick kind, if gained pcr amplification product is 252bp base fragments, paddy rice to be measured is rice blast susceptible variety.
Further, in step (2), the 272bp base fragments are SEQ ID NO：Nucleotide sequence shown in 1, institute
252bp base fragments are stated for SEQ ID NO：Nucleotide sequence shown in 2.
Further, in step (1), the extracting method of the genomic DNA extracted from paddy rice to be measured is alkaline-heating method, specifically
Step is：Take 1/10 rice grain endosperm, add 40ml 0.2Mol/L sodium hydrate aqueous solution, under the conditions of 100 DEG C
Water-bath 3 minutes, adds 60ml 0.17Mol/L tris-HCL, water-bath 1 minute under the conditions of 100 DEG C.
Further, in step (1), the reaction system of PCR amplifications is：Containing Mg2+The μ l of 10X Buffer 2,10mM's
DNTP 0.4 μ l, 5 μM such as SEQ ID NO：Nucleotide sequence shown in 3 and 5 μM such as SEQ ID NO：Nucleosides shown in 4
Each 2 μ l of acid sequence, μ l, the 5U/ μ l of genomic DNA 1 extracted from the paddy rice to be measured μ l of taq enzymes 0.5, remaining is ultra-pure water, instead
It is 20 μ l to answer volume, is added dropwise one and drips mineral oil covering；PCR amplification response procedures be：94℃ 5min；94 DEG C of 60s, 53.5 DEG C
60s, 72 DEG C of 60s, 35 circulations；72℃10min.
The present invention also provides above-mentioned molecular labeling, primer pair and detects the method for Rice Resistance To Rice Blast in rice breeding
In application.
Beneficial effects of the present invention are embodied in：
Molecular labeling of the present invention is located at resistant gene Pigm gene internal, closely connects with resistance gene of rice blast
Lock, compared with non-genomic inner marker, molecular labeling of the present invention is not in separation, can more effectively improve breeding efficiency.
Primer pair of the present invention can be expanded by the gene extracted from paddy rice to be measured obtained by simple process well
Group, overcoming other molecular labelings in the prior art with the gene close linkage can not expand by obtained by simple process
The problem of genome extracted from paddy rice to be measured, the method for obtaining present invention detection Rice Resistance To Rice Blast, this method can
Rapid gene evaluation and screening is carried out to the rice seed grain before sowing, then selectively sows, reduces recruitment land used, shortening is educated
In the cycle of kind, further improve breeding efficiency.
The method of present invention detection Rice Resistance To Rice Blast is overcome in conventional breeding because Phenotypic Selection is easily by environmental factor
Disturb and cause the low problem of breeding efficiency, also overcome in molecular mark, due to molecular labeling and blast resisting
Gene has certain genetic distance, the problem of there is a possibility that separation in breeding process, present invention detection rice blast
The method of resistance carries out early generation selection to resistance gene of rice blast using primer pair of the present invention and shortens breeding cycle,
So as to filter out the rice material of blast resisting quickly.Present invention can apply to rice breeding, to improve breeding efficiency and water
Rice yield level.
Brief description of the drawings
Fig. 1 is to use SEQ ID NO：Nucleotide sequence and SEQ ID NO shown in 5：What the nucleotide sequence shown in 6 was constituted
After primer pair amplifies disease-resistant variety paddy plum 4 and not anti-kind FD1715, amplified production base fragment comparison diagram.
Fig. 2 is SEQ ID NO：Nucleotide sequence and SEQ ID NO shown in 3：What the nucleotide sequence shown in 4 was constituted draws
Thing to expand not anti-kind FD1715, disease-resistant variety paddy plum 4 and and both cenospecies electrophoretogram.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used in following embodiments, unless otherwise specified, by normal
Rule biochemical reagents company is commercially available.
Embodiment 1
Rice blast resistance Characters Identification special primer pair SEQ ID NO：Nucleotide sequence and SEQ ID NO shown in 3：4
The acquisition of shown nucleotide sequence
1.1 parents' genome amplifications
Parents for creating paddy rice RIL (recombinant inbred line, RIL) colony are paddy respectively
Plum 4 and the FD1715 of Anhui Feng great Zhong industry limited company seed selection.FD1715 is female parent, not blast resisting；Paddy plum 4 is father
This, high resistant to rice blast.
The genomic DNA of parents' blade is extracted using CTAB methods, with Yiwen Deng (Yiwen Deng, et al.
Epigenetic regulation of antagonistic receptors confers rice blast resistance
with yield balance[J].2017.355(6328):962) sequence announced, designs primer, by SEQ ID NO：5 institutes
The nucleotide sequence and SEQ ID NO shown：Nucleotide sequence composition shown in 6, after the synthesis of commission Beijing Zi Xi biotech firms, enters
Performing PCR amplification experiment.PCR amplification reaction system be：The μ l of 10X Buffer 2 (contain Mg2+), the μ l (10mM) of dNTP 0.4,
SEQ ID NO：Nucleotide sequence and SEQ ID NO shown in 5：Each 2 μ l of nucleotide sequence (5 μM) shown in 6, genomic DNA
1 μ l (40ng/ μ l), the μ l of taq enzymes 0.5 (5 υ/μ l), remaining is ultra-pure water, and reaction volume is 20 μ l, and a drop mineral oil is added dropwise and covers
Lid.PCR amplification response procedures be:94℃5min；94 DEG C of 60s, 53.5 DEG C of 60s, 72 DEG C of 60s, 35 circulations；72℃
10min。
Pcr amplification product sends to sequencing, and sequencing company is Beijing Zi Xi biotech firms, and sequencing result exists in NCBI websites
Line comparison result is shown in Fig. 1.Dashed region represents 20bp missing in figure, and wherein query is the male parents of Gu Mei 4, high resistant to rice blast
Sequence；Sbjct is FD1715 maternal, not the sequence of blast resisting.
1.2 primers are synthesized and verified
According to Fig. 1 sequencing comparison result, there is 20bp insertion and deletions diff area to design primer across two sequences, on
Trip primer is SEQ ID NO：Nucleotide sequence shown in 3, anti-sense primer is SEQ ID NO：4 shown nucleotide sequence.Committee
Hold in the palm the synthesis of Beijing Zi Xi biotech firms.Sense primer and anti-sense primer (primer pair is named as into QK1) are located in gene Pigm
Portion's functional area.
Utilize the primer amplification paddy plum 4 newly synthesized and FD1715, and both cenospecies.Using alkaline-heating method from water to be measured
Genomic DNA is extracted in rice, is concretely comprised the following steps：Take 1/10 rice grain endosperm, add 40ml 0.2Mol/L hydroxide
Sodium water solution, water-bath 3 minutes under the conditions of 100 DEG C, adds 60ml 0.17Mol/L tris-HCL, in 100 DEG C of conditions
Lower water-bath 1 minute, that is, extract the genomic DNA for obtaining paddy rice to be measured, and 4 DEG C of refrigerator coolings are standby.
PCR amplification reaction system be：The μ l of 10X Buffer 2 (contain Mg2+), the μ l (10mM) of dNTP 0.4, sense primer and
Each 2 μ l of anti-sense primer (5 μM), the μ l (5U/ μ l) of 1 μ l, taq enzyme of genomic DNA 0.5 extracted from paddy rice to be measured, remaining is
Ultra-pure water, reaction volume is 20 μ l, is added dropwise one and drips mineral oil covering.PCR amplification response procedures be： 94℃5min；94℃
60s, 53.5 DEG C of 60s, 72 DEG C of 60s, 35 circulations；72℃10min.
6% Polyacrylamide Gel Electrophoresis of pcr amplification product, rapid silver staining is dyed and observed and takes pictures, and sees figure
2, FD1715, Gu Mei 4 and each 2 repetitions of each material of cenospecies in figure, Marker sizes are 250bp.Pcr amplification product is entered
Row sequencing, can be clearly seen that, Gu Mei 4 has an electrophoresis band from the electrophoretogram of amplified production, through the electrophoresis band pair is sequenced
The amplified production answered is 272bp base fragments, is SEQ ID NO：Nucleotide sequence shown in 1；FD1715 has an electrophoresis
Band, is 252bp base fragments through the corresponding amplified production of the electrophoresis band is sequenced, is SEQ ID NO：Nucleotides sequence shown in 2
Row；Hybrid has two electrophoresis bands, is 272bp and 252bp base pieces through the corresponding amplified production of the electrophoresis band is sequenced
Section, is SEQ ID NO：Nucleotide sequence and SEQ ID NO shown in 1：Nucleotide sequence shown in 2.
Embodiment 2
QK1 amplified production and the relevance verification of Rice Resistance To Rice Blast character
With high resistant to rice blast material paddy plum 4 and blast resisting material FD1715 is not parent, both hybridize after 432 F2
It is subjects for material.Genomic DNA is extracted from paddy rice to be measured using alkaline-heating method, specific steps with embodiment 1.Carry out
PCR amplification experiments.PCR reaction systems are：The μ l of 10X Buffer 2 (contain Mg2+), dNTP 0.4 μ l (10mM), SEQ ID NO：3
Shown nucleotide sequence and SEQ ID NO：Each 2 μ l of nucleotide sequence (5 μM) shown in 4, the base extracted from paddy rice to be measured
Because of the μ l (5U/ μ l) of 1 μ l, taq enzymes of group DNA 0.5, remaining is ultra-pure water, and reaction volume is 20 μ l, and a drop mineral oil is added dropwise and covers
Lid.PCR amplification response procedures be：94℃5min；94 DEG C of 60s, 53.5 DEG C of 60s, 72 DEG C of 60s, 35 circulations；72℃
10min。
6% Polyacrylamide Gel Electrophoresis of pcr amplification product, rapid silver staining is dyed and observed and photographs to record,
Wherein 105 plants of the 272bp of homozygosis, 97 plants of homozygosis 252bp, 220 plants of heterozygosis banding pattern.
Indoor inoculation material includes FD1715, Gu Mei No. 4 numbers, both hybrid strains.It is seeded in respectively after seed presoaking and germinating
32 hole plastics connect in version, seedling medium be pot flowers culture medium, seedling in 26 DEG C -28 DEG C of artificial climate room 12h illumination,
12h dark culturings to three arrive four leaf stage.Using single biological strain about 1 × 105The rice blast fungus spore suspension live body of/ml concentration
Spray inoculation, in after 26 DEG C of dark moisturizing 24h, continues to keep illumination high humidity 5-7d, with reference to Bonman 0-5 levels standard survey disease
Feelings (0-2 grades are disease-resistant, and 3-5 grades are susceptible).
It is analyzed as follows：It is as shown in table 1 below, for there is 105 plants of homozygosis 272bp in examination paddy rice sample, wherein 102 plants of performances
To be disease-resistant, have 3 plants it is susceptible；And it is susceptible without 92 plants in 97 materials of 272bp fragments, have 5 plants it is disease-resistant；220 plants of heterozygosis,
216 plants show as disease-resistant, and 4 plants susceptible；Both uniformity reach 97.1%.It was therefore concluded that, SEQ ID NO：3
Shown nucleotide sequence and SEQ ID NO：Shown in 4 nucleotide sequence composition QK1 primer pairs be and rice blast resistance
The molecular labeling of shape related locus phase close linkage, available for rice molecular assistant breeding.
The QK1 amplified productions qualification result of table 1 is compareed with field rice blast inoculation test
It should be understood that example as described herein and embodiment are not intended to limit the invention, this area only for explanation
Technical staff can make various modifications or change according to it, within the spirit and principles of the invention, any modification for being made,
Equivalent substitution, improvement etc., should be included in the scope of the protection.
。
SEQUENCE LISTING
Sequence table
<110>Anhui Feng great Zhong industry limited company
<120>A kind of molecular labeling, primer and its application with resistance gene of rice blast close linkage
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<170>PatentIn version 3.1
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tatcggctaa gacagaatat cagacctaac taaaccgatg cgtctctaaa cacaatgcaa 180
ttaattagag atataattga gatatcagct aggcaaatat atcaaccaaa ctggagcgat 240
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cagacctaac taatgggatg cgtctctaaa cacaatgcaa ttaattagag atataattga 180
gatatcagct aggcaaatat atcaaccaaa ctggagcgat ccaagagatc ggagcaatgc 240
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Claims (9)
1. a kind of and resistance gene of rice blast close linkage molecular labeling, it is characterised in that the core of the molecular labeling
Nucleotide sequence such as SEQ ID NO：1 shown or SEQ ID NO：Shown in 2.
2. a kind of primer pair for expanding molecular labeling as claimed in claim 1, it is characterised in that including primer 1 and primer 2,
The nucleotide sequence of the primer 1 such as SEQ ID NO：Shown in 3, the nucleotide sequence such as SEQ ID NO of the primer 2：4 institutes
Show.
3. application of the molecular labeling as claimed in claim 1 in rice breeding.
4. application of the primer pair as claimed in claim 2 in rice breeding.
5. a kind of method for detecting Rice Resistance To Rice Blast, it is characterised in that comprise the following steps：
(1) PCR is expanded：Using the genomic DNA that is extracted from paddy rice to be measured as DNA cloning template, with SEQ ID NO：Shown in 3
Nucleotide sequence and SEQ ID NO：Nucleotide sequence composition shown in 4 is amplimer to entering performing PCR amplification；
(2) identification of amplified production：If gained pcr amplification product is 272bp base fragments, paddy rice to be measured is blast resisting product
Kind, if gained pcr amplification product is 252bp base fragments, paddy rice to be measured is rice blast susceptible variety.
6. the method for Rice Resistance To Rice Blast is detected as claimed in claim 5, it is characterised in that described in step (2)
272bp base fragments are SEQ ID NO：Nucleotide sequence shown in 1, the 252bp base fragments are SEQ ID NO：Shown in 2
Nucleotide sequence.
7. the method for the detection Rice Resistance To Rice Blast as described in claim 5 or 6, it is characterised in that in step (1), from treating
The extracting method for surveying the genomic DNA extracted in paddy rice is alkaline-heating method, is concretely comprised the following steps：Take 1/10 rice grain endosperm, plus
Enter 40ml 0.2Mol/L sodium hydrate aqueous solution, water-bath 3 minutes under the conditions of 100 DEG C add 60ml 0.17Mol/L
Tris-HCL, water-bath 1 minute under the conditions of 100 DEG C.
8. the method for the detection Rice Resistance To Rice Blast as described in claim 5 or 6, it is characterised in that in step (1), PCR expands
The reaction system of increasing is：Containing Mg2+10X Buffer 2 μ l, 10mM dNTP 0.4 μ l, 5 μM such as SEQ ID NO：Shown in 3
Nucleotide sequence and 5 μM such as SEQ ID NO：Each 2 μ l of nucleotide sequence shown in 4, the gene extracted from paddy rice to be measured
Group DNA 1 μ l, the 5U/ μ l μ l of taq enzymes 0.5, remaining is ultra-pure water, and reaction volume is 20 μ l, is added dropwise one and drips mineral oil covering；
PCR amplification response procedures be：94℃5min；94 DEG C of 60s, 53.5 DEG C of 60s, 72 DEG C of 60s, 35 circulations；72℃10min.
9. application of the method for the detection Rice Resistance To Rice Blast as any one of claim 5 to 8 in rice breeding.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628627A (en) * | 2018-12-11 | 2019-04-16 | 华智水稻生物技术有限公司 | The SNP marker development and application of broad-spectrum rice-blast resistant gene of paddy rice Pigm |
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| CN106148510A (en) * | 2016-06-16 | 2016-11-23 | 淮阴师范学院 | Resistance gene of rice blast Pi5 specific Function molecular marker and application thereof |
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| CN102899400A (en) * | 2012-09-05 | 2013-01-30 | 袁隆平农业高科技股份有限公司 | Molecule marking method for rice anti-rice blast gene Pigm |
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