CN109468400A - Rice blast resistant gene Pi36 codominant marker and application - Google Patents
Rice blast resistant gene Pi36 codominant marker and application Download PDFInfo
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Abstract
The present invention provides rice blast resistant gene Pi36 codominant marker, including label SNP1 and/or label SNP2, wherein polymorphic site contained by label SNP1 is located at corresponding at No. 8 2886707 bases of chromosome of rice OryzasativaLcv.Nipponbare, and base is G or A at this；Polymorphic site contained by SNP2 is marked to be located at corresponding at No. 8 2888118 bases of chromosome of rice OryzasativaLcv.Nipponbare, base is G or A at this.Specificity amplification primer is separately designed for the molecular labeling, the detection of Pi36 genotype is carried out by PCR amplification.Molecular labeling of the invention is codominance, can effectively distinguish 3 kinds of different genotypes, the molecular labeling can be used for breeding anti-rice blast rice, shorten the breeding cycle of anti-rice blast rice, accelerate breeding speed, reduces breeding cost, it is low in cost with easy to operate, the advantages that period is short.
Description
Technical field
The invention belongs to molecular biology and plant molecular breeding fields, specifically, being related to rice blast resistant gene
Pi36 codominant marker and application.
Background technique
Rice is as China main food industrial crops, and throughout the year by the threat from pest and disease damage, wherein rice blast is danger
One of evil disease the most serious.In each rice producing region of China, rice blast has generation every year, and yield can subtract when disease pest is prevailing
Few 10%~20%, more serious area is up to 40%~50%, or even total crop failure.Largely it can be relieved rice blast using blast resisting medicament
The harm of disease but the cost for also having increased considerably Rice Production, and environment is polluted, practice have shown that breeding Varieties Resistant To Rice Blast is
Control rice blast measure the most cost-effective.But since rice blast biological strain is numerous, variation is fast, that often newly releases is disease-resistant
Kind loses resistance just shortly after in plantation to reduce utility value.Therefore different blast resistant genes are excavated and utilize,
Cultivating, there are the rice varieties of different disease-resistant genes to play a significant role to breeding wide spectrum, resistance to rice blsat kind.
In conventional breeding for disease resistance, Resistance Identification is cumbersome, need to put into more manpower and cost, identification are influenced vulnerable to external condition
And cause result inaccurate, therefore breeding efficiency is relatively low.Molecular mark is that a kind of utilize closely connects with functional gene
Functional label in lock or gene combines genotype and table identification to carry out the modern breeding skill of selection to target shape in offspring
Art.This method can greatly shorten the breeding time limit, improve breeding efficiency, and also a save a large amount of human and material resources cost.Xian
Rice varieties Kasalath shows stable, high-caliber resistance to the part rice blast biological strain of isolated in China.Pass through figure position
Cloning process has been cloned rice blast resistance gene Pi36 (GenBank:DQ900896.1) from kasalath, and Pi36 is located at the 8th
The short arm of a chromosome is the single copy gene of a constitutive expression, encodes the protein product of 1056 amino acid.Disease-resistant gene
The clone of Pi36 is that exploitation Pi36 gene function label carries out molecular mark, and further breeding has different anti-rice
The kind of seasonal febrile diseases level is of great significance.
Summary of the invention
The object of the present invention is to provide rice blast resistant gene Pi36 codominant marker and applications.
In order to achieve the object of the present invention, in a first aspect, the present invention provides rice blast resistant gene Pi36 co-dominant molecular
It marks (Pi36-RS), including label SNP1 and/or label SNP2, wherein polymorphic site contained by label SNP1 is located at corresponding
At No. 8 2886707 bases of chromosome of rice OryzasativaLcv.Nipponbare, base is G or A at this；Mark polymorphic position point contained by SNP2
In corresponding at No. 8 2888118 bases of chromosome of rice OryzasativaLcv.Nipponbare, base is G or A at this.
Wherein, rice OryzasativaLcv.Nipponbare genome version number is IRGSP1.0.
Preferably, marking the nucleotide sequence of SNP1, (the 20th bit base is SNP site, n g as shown in SEQ ID NO:1
Or a), marking the nucleotide sequence of SNP2, (the 46th bit base is SNP site, and n is for g or a) as shown in SEQ ID NO:2.
Second aspect, the present invention is provided to detect the ARMS-PCR primer of the molecular labeling, wherein for detecting mark
The primer for remembering SNP1 includes Pi36-RR and Pi36-RF, and nucleotide sequence is as shown in SEQ ID NO:3-4；
Primer for detecting label SNP2 includes Pi36-SF and Pi36-SR, nucleotide sequence such as SEQ ID NO:5-
Shown in 6.
The third aspect, the present invention provide the inspection containing primer Pi36-RR/Pi36-RF and primer Pi36-SF/Pi36-SR
Test agent or kit.
Fourth aspect, the present invention provide the molecular marker SNP 1, SNP2, primer Pi36-RR/Pi36-RF, primer
Following any application of Pi36-SF/Pi36-SR or detection reagent or kit containing the primer:
1) application in the genotype identification of rice blast resistant gene Pi36；
2) identifying and screening the application in anti-rice blast rice germ plasm resource；
3) application in anti-rice blast rice kind molecular mark.
5th aspect, the present invention provide the methods of genotyping of rice blast resistant gene Pi36, include the following steps:
(1) genomic DNA of rice to be measured is extracted；
(2) genomic DNA obtained using step (1) utilizes primer Pi36-RR/Pi36-RF and primer Pi36- as template
SF/Pi36-SR carries out PCR amplification；
(3) amplified production is analyzed.
Method above-mentioned, step (2) PCR reaction system are as follows: 1ul 10 × PCR reaction buffer, 0.8ul 10mM
DNTP, 10uM each primer 0.15ul, 2.5U/ul Taq archaeal dna polymerase 0.1ul, 2ul DNA profiling, ddH2O is mended to 10ul.
PCR reaction condition are as follows: 95 DEG C initial denaturation 5 minutes；95 DEG C are denaturalized 30 seconds, 56 DEG C -58 DEG C (preferably 56 DEG C) annealing 45
Second, 72 DEG C extend 45 seconds, total 30-35 circulation (preferably 32 circulations)；72 DEG C extend 5 minutes.
Preferably, step (3) analyzes amplified production using agarose gel electrophoresis.
Step (3) specifically: if the characteristic bands of 345bp occurs in amplified production, determine that rice to be measured is anti-for Pi36
Disease allele is homozygous；If the characteristic bands of 433bp occurs in amplified production, determine rice to be measured for the susceptible equipotential of Pi36
Gene pure type；If two kinds of banding patterns of 345bp and 433bp occurs in amplified production, determine rice to be measured for heterozygous genotypes.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
(1) the present invention is based on Pi36 gene internal sequence differences to design molecular labeling, and there is no heredity exchange, accuracys
It is high.
(2) the amplified production difference of molecular labeling of the present invention is obvious, can be directly used for agarose gel electrophoresis detection, side
Just quick, it is low in cost.
(3) molecular labeling of the invention is codominance, can effectively distinguish 3 kinds of different genotypes, this method can be used for breeding
Anti-rice blast rice shortens the breeding cycle of anti-rice blast rice, accelerates breeding speed, reduces breeding cost, has operation letter
It is single, low in cost, the advantages that period is short.
Detailed description of the invention
Fig. 1 is the known Pi36 gene order ratio containing blast resisting or the rice varieties for feeling rice blast Pi36 allele
To figure.Wherein, Kasalath is the known rice varieties containing the disease-resistant allele of Pi36 in A and B, 9311, Nipponbare,
IR64, another name for Sichuan Province extensive 498,02428 are the rice varieties of the known susceptible allele containing Pi36.
Fig. 2 is the detection electrophoretogram of rice breed in the embodiment of the present invention 4.Wherein, M:DL1000DNA marker,
Wherein swimming lane 1-26 be corresponding in turn to accounted for for kasalath, China, 638S, Feng Yuzhan, salt rice 1531, Hua Hui 284, Cheng Hui 19, China Resources 2
Number, Mianhui 3728, Mf63, Shanghai round-grained rice 5,02428, Shanghai round-grained rice 6, poplar round-grained rice 5507, hot round-grained rice 35, Yu Zhenxiang, more round-grained rice 0618, emblem round-grained rice 602, town
Rice 819, distant salt 287, Jiangsu round-grained rice 2, land reclamation and cultivation 31, Fukuniski, Zhejiang round-grained rice 75, non-irrigated two excellent No. 1, Anhui two excellent 385.
Fig. 3 is the electrophoretogram in the embodiment of the present invention 5 to F2 crowd surveillance.Wherein, M:DL1000DNA marker, swimming lane
1,2 be respectively disease-resistant parent kasalath and Susceptible parent 9311, and swimming lane 3-38 is the F2 group constructed using kasalath/9311
Body single plant, each affiliated genotype mark of material is above corresponding swimming lane, and S represents susceptible allelic gene type, and R represents disease-resistant equipotential
Gene type, H represent hybrid type.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW,
Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
The exploitation of 1 Pi36 gene molecule marker of embodiment
Rice blast resistant gene Pi36 is located at No. 8 the short arm of a chromosome of rice, by Kasalath, 9311, OryzasativaLcv.Nipponbare
Deng known blast resisting or sense rice blast rice kind Pi36 genome area carries out sequencing and ncbi database compares online
Analysis finds two specific SNP sites, and wherein SNP1 is located at No. 8 2886707 bases of chromosome of OryzasativaLcv.Nipponbare sequence, Pi36
Disease-resistant equipotential is G, and susceptible equipotential is A (as shown in Figure 1A)；SNP2 is located at No. 8 chromosomes 2888118 of OryzasativaLcv.Nipponbare sequence, Pi36
Disease-resistant equipotential is G, and susceptible equipotential is A (as shown in Figure 1B).For this at two SNP site develop two couples of dominant markers, use respectively
In identifying disease-resistant equipotential and susceptible equipotential, two couples of dominant markers, which are applied in combination, can be realized the identification of Pi36 different genotype.
The detection primer of 2 Pi36 gene codominant marker of embodiment designs
Detection primer, design of primers principle are designed for two Pi36 gene specific molecular labelings that embodiment 1 is developed
It is as follows.
Firstly, design expands the specific primer of disease-resistant allele, with the complementary base of the disease-resistant equipotential G base in the site SNP1
Base C holds design primer Pi36-RR as the 3 ' of reverse primer, and C base is changed to A base at the 3rd bit base at 3 ' ends and is drawn
Enter mispairing, then designs matching forward primer Pi36-RF, primer Pi36-RR and Pi36-RF (SEQ ID at its upstream
NO:3-4) can only the disease-resistant equipotential of specific amplified Pi36, generate a 345bp band, and the susceptible equipotential of Pi36 in amplification without band；
Then, design expands the specific primer of susceptible allele, using the susceptible equipotential A in the site SNP2 as 3 ' end designs
Forward primer Pi36-SF, and go out in 3 ' the 3rd bit bases and A base is changed to the introducing mispairing of T base, then designing downstream therewith
Matched reverse primer Pi36-SR, primer Pi36-SF and Pi36-SR (SEQ ID NO:5-6) can only specific amplified Pi36 it is susceptible
Equipotential, generates the band of a 433bp, and without band when the disease-resistant equipotential amplification of Pi36.
Primer sequence is as shown in table 1, and four primers are used in mixed way the codominant marker detection primer group of composition Pi36,
When amplified production only has 345bp band, rice to be measured carries the disease-resistant allele of Pi36；When amplified production only has 433bp item
When band, rice to be measured carries the susceptible allele of Pi36；When amplified production has two kinds of banding patterns of 345bp and 433bp, rice to be measured
For Pi36 heterozygous genotypes rice.
1 Pi36 specific molecular marker primer sequence of table and relevant parameter
The foundation of 3 rice blast resistant gene Pi36 specific molecular marker detection method of embodiment
According to the detection primer for two molecular labelings of Pi36 that embodiment 2 designs, the response procedures and reactant of PCR are designed
System determines following response procedures and system by continuing to optimize:
PCR reaction system (10 μ L): 1ul 10 × PCR reaction buffer, 0.8ul 10mM dNTP, 4 primers (10 μM)
It is 0.15 μ L, 0.1ul Taq archaeal dna polymerase (2.5U/ul), 2ul DNA profiling, ddH2O is mended to 10ul.
PCR reaction condition are as follows: 95 DEG C initial denaturation 5 minutes；95 DEG C are denaturalized 30 seconds, and 56 DEG C are annealed 30 seconds, and 72 DEG C extend 45 seconds,
Totally 32 circulations；72 DEG C extend 8 minutes.
4 rice blast resistant gene Pi36 specific molecular marker of embodiment is in detection paddy disease-resistant and susceptible variety
Using
1, rice material
Pi36 donor parents Kasalath and 25 parts of breeding parent materials or commercially available rice varieties, specifically include: China accounts for,
Beautiful needle perfume, 638S, Feng Yuzhan, Hua Hui 284, Cheng Hui 19, China Resources 2, Mianhui 3728, Mf63, Shanghai round-grained rice 5,02428, Shanghai round-grained rice 6, poplar
Round-grained rice 5507, hot round-grained rice 35, more round-grained rice 0618, emblem round-grained rice 602, town rice 819, distant salt 287, Jiangsu round-grained rice 2, salt rice 1531, land reclamation and cultivation 31,
Fukuniski, Zhejiang round-grained rice 75, non-irrigated two excellent No. 1, Anhui two excellent 385.
2, oryza sativa genomic dna extracts and primer synthesizes
Using CTAB method extract above-mentioned rice material genomic DNA and synthesis table 1 shown in primer sequence.
3, PCR is detected
The reaction system and response procedures of PCR is as described in Example 3.Amplified production electrophoresis in 2% Ago-Gel is used
Gel imager scanning record result.
4, interpretation of result
The electrophoresis result of pcr amplification product is as shown in Fig. 2, 1 swimming lane Pi36 donor material Kasalath specific amplification goes out
The band of 345bp, the corresponding parent material of 2-26 swimming lane amplify the band of 433bp.In order to verify the result of PCR detection
The corresponding molecular labeling target area of Kasalath and 25 parent material has been carried out sequencing and compared by accuracy.The result shows that point
Son label testing result is consistent with sequencing result, it was demonstrated that primer provided by the invention being capable of precise Identification blast resistant gene Pi36
Genotype, it can be achieved that the variety resources of rice of precise and high efficiency screening.
The detection that 5 rice blast resistant gene Pi36 of embodiment is separated in the single-gene of F2 group
1, rice material
Disease-resistant parent kasalath and Susceptible parent 9311, and the random choosing in the F2 group of two parents building
The 96 F2 single plants taken.
2, the extraction of oryza sativa genomic dna and PCR detection
The primer of the extraction of oryza sativa genomic dna and PCR detection, response procedures and system are as described in Example 4.
3, interpretation of result
The electricity of the pcr amplification product of blast resisting parent Kasalath and sense rice blast parent 9311 and part F2 strain
For result of swimming as shown in figure 3, swimming lane 1,2 is respectively disease-resistant parent Kasalath and Susceptible parent 9311, swimming lane 3-38 is to utilize two
The part F2 single plant of parent's building randomly selected, below corresponding swimming lane, S represents susceptible etc. each affiliated genotype mark of material
Position gene type, R represent disease-resistant allelic gene type, and H represents hybrid type.The result shows that 96 F2 single plants are detected,
The segregation ratio of 3 kinds of different genotypes is 25SS:50H:21RR, and Mendel's single-gene segregation ratio of 1:2:1 is met through Chi-square Test
(χ2=0.50 is less than χ2 0.05=5.99) it, therefore, should be labeled as codominant marker, it can be by two kinds of different homozygotes and heterozygosis
Sub- genotype distinguishes, and detection site shows as single-gene separation simultaneously.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Yuanlongping Agricultural Hi-Tech Co., Ltd., grand Co., Ltd, flat high-tech Zhong Ye research institute lake, Hunan
South Asia Hua Zhongye research institute
<120>rice blast resistant gene Pi36 codominant marker and application
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Claims (10)
1. rice blast resistant gene Pi36 codominant marker, which is characterized in that including label SNP1 and/or label
SNP2, wherein polymorphic site contained by label SNP1, which is located at, to be corresponded at No. 8 2886707 bases of chromosome of rice OryzasativaLcv.Nipponbare,
Base is G or A at this；It marks polymorphic site contained by SNP2 to be located at and corresponds to No. 8 2888118 alkali of chromosome of rice OryzasativaLcv.Nipponbare
Ji Chu, base is G or A at this.
2. molecular labeling according to claim 1, which is characterized in that mark the nucleotide sequence such as SEQ ID NO of SNP1:
Shown in 1, mark the nucleotide sequence of SNP2 as shown in SEQ ID NO:2.
3. the ARMS-PCR primer for detecting molecular labeling described in claim 1, which is characterized in that for detecting label SNP1
Primer include Pi36-RR and Pi36-RF, nucleotide sequence is as shown in SEQ ID NO:3-4；
Primer for detecting label SNP2 includes Pi36-SF and Pi36-SR, nucleotide sequence such as SEQ ID NO:5-6 institute
Show.
4. detection reagent or kit containing primer described in claim 3.
5. detection reagent described in primer described in molecular labeling as claimed in claim 1 or 2, claim 3 or claim 4 or reagent
Following any application of box:
1) application in the genotype identification of rice blast resistant gene Pi36；
2) identifying and screening the application in anti-rice blast rice germ plasm resource；
3) application in anti-rice blast rice kind molecular mark.
6. the methods of genotyping of rice blast resistant gene Pi36, which comprises the steps of:
(1) genomic DNA of rice to be measured is extracted；
(2) genomic DNA obtained using step (1) carries out PCR amplification using primer described in claim 3 as template；
(3) amplified production is analyzed.
7. according to the method described in claim 6, it is characterized in that, PCR reaction system used by step (2) are as follows: 1ul 10
× PCR reaction buffer, 0.8ul 10mM dNTP, 10uM each primer 0.15ul, 2.5U/ul Taq archaeal dna polymerase 0.1ul,
2ul DNA profiling, ddH2O is mended to 10ul.
8. according to the method described in claim 6, it is characterized in that, PCR reaction condition used by step (2) are as follows: 95 DEG C pre-
Denaturation 5 minutes；95 DEG C are denaturalized 30 seconds, and 56 DEG C -58 DEG C are annealed 45 seconds, and 72 DEG C extend 45 seconds, total 30-35 circulation；72 DEG C extend 5
Minute.
9. according to the method described in claim 6, it is characterized in that, step (3) is produced using agarose gel electrophoresis analysis amplification
Object.
10. the method according to claim 6, which is characterized in that step (3) specifically: if amplified production
There are the characteristic bands of 345bp, then determines that rice to be measured is homozygous for the disease-resistant allele of Pi36；If amplified production occurs
The characteristic bands of 433bp then determine that rice to be measured is homozygous for the susceptible allele of Pi36；If there is 345bp in amplified production
With two kinds of banding patterns of 433bp, then determine rice to be measured for heterozygous genotypes.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1821406A (en) * | 2006-03-06 | 2006-08-23 | 华南农业大学 | Resistance gene Pi 36 of rice blast and its use |
| CN106755475A (en) * | 2017-01-17 | 2017-05-31 | 中国水稻研究所 | A kind of method of early 39 rice blast resistance genes in identification super early rice |
-
2018
- 2018-12-07 CN CN201811495414.0A patent/CN109468400B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1821406A (en) * | 2006-03-06 | 2006-08-23 | 华南农业大学 | Resistance gene Pi 36 of rice blast and its use |
| CN106755475A (en) * | 2017-01-17 | 2017-05-31 | 中国水稻研究所 | A kind of method of early 39 rice blast resistance genes in identification super early rice |
Non-Patent Citations (2)
| Title |
|---|
| XINQIONG LIU等: "the in silico map-based cloning of pi36, a rice coiled-coil-nucleotide-binding site-leucine-rich repeat gene that confers race-specific resistance to the blast fungus", 《GENETICS》 * |
| 王丽丽等: "辽宁地区水稻资源抗稻瘟病基因的检测分析", 《植物遗传资源学报》 * |
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