CN109628627A - The SNP marker development and application of broad-spectrum rice-blast resistant gene of paddy rice Pigm - Google Patents
The SNP marker development and application of broad-spectrum rice-blast resistant gene of paddy rice Pigm Download PDFInfo
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Abstract
The present invention provides a kind of and blast resistant gene Pigm close linkage SNP marker K_060508, it is G or A that the SNP marker, which detects base at the 10421726th site of No. 6 chromosome of rice, and the primer sets of the SNP marker based on KASP technological development are combined into Primer X, Primer Y and Primer C.The present invention carries out gene rapid typing using KASP technology pair and the SNP marker of blast resistant gene Pigm close linkage, can applying in commercialization molecular breeding with high, medium and low flux；Develop the efficiency of selection of SNP marker phenotype simultaneously and reach 100%, can in the different germ plasm resources such as long-grained nonglutinous rice, japonica rice fast and accurate detection wide spectrum blast resistant gene Pigm；And the red tapes such as digestion, electrophoresis and sequencing are not needed in the detection process, reduce the use of the Toxics such as pollution and the EB (ethidium bromide) of aerosol, it can be in the molecular marker assisted selection of breeding early stage progress prospect, reduce the field planting scale of breeding population, breeding cost is reduced, breeding process is accelerated.
Description
Technical field
The present invention relates to molecular labeling and crop breeding fields, specifically, being related to broad-spectrum rice-blast resistant gene of paddy rice Pigm
SNP marker development and application.
Background technique
The rice blast as caused by Pyricularia oryzae (Magnapotheoryzae) is that a kind of global rice makees disease, and the whole world is every
Year because for Rice Yield Loss Caused up to 11%~30%, long-term practice shows that cultivating disease-resistant variety (being) is mesh caused by rice blast
The most economical effective measures of preceding prevention and treatment rice blast.In recent decades, with the development of molecular genetics, molecular marker assisted selection
Very important effect is played in paddy disease-resistant breeding, contains target gene using efficient molecular marker assisted selection
Single plant is hybridized, and can precisely carry out the breeding of objective trait, and can reduce breeding population field planting scale, shortens breeding
The time limit saves breeding cost.
Currently, by genetic analysis, it is identified in rice to have named about 100 rice blast resistance genes, wherein 26
A successful clone (Liuetal., 2014).Pigm gene is related to leaf blast in rice resistance, is the anti-pest gene of wide spectrum, than public affairs
The anti-spectrum of broad-spectrum resistance gene Pi1, Pi2, Pi3 for recognizing are wider, and donor paddy plum No. 4 is to collected from the 30 of various regions and nations
29 performance highly resistances or immune in a strong pathogenic strain.Pigm is located in rice No. 6 chromosomal marker C5483 and C0428
Between in the section 70Kb, isolated with label S29742, C24901, PC22705, the base comprising 13 NBS-LRR class disease-resistant genes
Because of cluster (Dengetal., 2006), wherein it is albumen PigmR and PigmS that 2 tools are functional.PigmS can be competed with PigmR
The broad spectrum resistance that heterodimer inhibits PigmR to mediate is formed, reduces the evolution selection pressure of pathogen, slows down pathogen
Pathogenic evolution to PigmR, therefore the disease-resistant of Pigm mediation has durable broad spectrum, is more potential rice blast resistance
Gene (Dengetal., 2017).
In addition, Pigm is located on the site Pi2/9, and Pi2, Pi9, Pi-zt, Piz, Pi50 multiple allele each other, still
Respective gene order and it is anti-spectrum there is some difference, to realize to Pigm utilization just need accurately by Pigm with it is other multiple
Allele distinguishes.But molecular labeling used in detection Pigm is mostly SSR, InDel or CAPS label, these labels at present
Effectively Pigm and other multiple alleles cannot be distinguished, and the donor and receptor of Pigm cannot be distinguished completely, detection knot
Fruit easily causes to judge by accident.In addition, the detection process of the labels such as SSR, InDel or CAPS needs digestion, gel electrophoresis, operation
Complexity, experimental cost is high, and detection flux is small, easily pollutes the environment, generates harm to human body.Develop Pigm specificity SNP
Molecular labeling and the detection architecture for establishing high-throughput environmental protection have important meaning to application of the promotion Pigm in Hybrid breeding in commercial system
Justice.
Summary of the invention
The invention discloses the molecular labeling of broad-spectrum rice-blast resistant gene of paddy rice Pigm a kind of and application, the molecular labelings
It is the SNP marker with the anti-rice blast gene Pigm close linkage of wide spectrum, which is based on KASP technological development, can high-throughput detection water
10421726th bit base of No. 6 chromosome of rice varieties OryzasativaLcv.Nipponbare genome.Present invention application KASP technology is to SNP molecule mark
Remember row Genotyping into, have many advantages, such as easy to operate, low in cost, detection cycle is short, label stability it is good, can accurately by
The blast resistant gene of Pigm and other close linkages or equipotential distinguish, and can accurately detect in low generation population
Pigm improves breeding efficiency, shortens the breeding time limit, is of great significance to the resistance of improvement rice blast.
Specifically, a kind of development process of the molecular labeling of rice blast resistance gene Pigm of the present invention is shown in Fig. 1.According to
Document report, Pigm gene are located at the section 10387509-10390465 of No. 6 chromosomes of rice, to this gene interval and its attached
The SNP site of nearly two sides is excavated.The SNP site picked out extracts flanking sequence, and utilizes online design of primers website
BatchPrimer3 carries out design of primers to it.Marked for these candidate SNPs, to Pigm genetic donor material paddy plum No. 4 and
Other 22 without Pigm rice varieties carry out KASP reaction verifying, pick out and effect is isolated and expanded with Pigm donor material
The good SNP marker K_060508 of fruit.We carry out the chain SNP marker of the resistant gene selected using about 190 parts of materials
Natural population's verifying, it was demonstrated that the Pigm gene loci that the present invention detects is the resistance locus of high specific.Utilize Pigm donor parent
The group offspring that this paddy plum No. 4 and CO39 hybridization generate has carried out phenotype verifying, demonstrates feasibility and standard of the invention again
True property.
In order to solve the problems in the existing technology, the object of the present invention is to provide tight with rice blast resistance gene Pigm
Close chain SNP (single nucleotide polymorphism) molecular labeling K_060508 and its application.
In order to achieve the object of the present invention, technical scheme is as follows:
A kind of SNP marker with blast resistant gene Pigm close linkage, the molecular labeling are and blast resisting
The SNP marker K_060508 that gene Pigm is isolated, the SNP marker detect the 10421726th site of No. 6 chromosome of rice
Place's base is G or A；
It is a kind of for detect molecular labeling described in claim 1 primer combine include: (1) two specific primer:
Primer X:5 '-CCAAGCAAATTACCACAACG-3 '；PrimerY:
5’-CCAAGCAAATTACCACAACA
GC-3'；(2) universal primers: Primer C:5 '-TTGTTATACGGTTTAATTAAGGTGA-3 ' is specifically shown in
Table 1.
1 flag sequence table of table
A kind of kit for the SNP marker detecting blast resistant gene Pigm, the kit includes as above-mentioned
Primer combination.
The molecular labeling is in detection blast resistant gene Pigm, in blast resistant gene Pigm assistant breeding and selecting
Educate the application in the rice pest insects of blast resisting.
Further, the application carries out Pigm parting including the use of KASP or Pigm is anchored in genetic chip.
Further, include the following steps:
S1, rice sample gene group DNA is extracted；
S2, using oryza sativa genomic dna as template, utilize above-mentioned primer combination carry out KASP reaction detection；Wherein, Liang Tiaote
Specific primer is separately connected different fluorescence sequences；
If S3, only detecting the corresponding fluorescence signal of the connected fluorescence sequence of primer PrimerY, detection site is base A,
Judge rice sample for the pure and mild type of carrying blast resistant gene Pigm；If only detecting the connected fluorescence sequence of primer Primer X
Corresponding fluorescence signal, then detection site is bases G, judges rice sample for the pure and mild type without blast resistant gene Pigm；
If being detected simultaneously by two kinds of fluorescence, judge rice sample for the heterozygous of blast resistant gene Pigm.
Further, 5 ' section of specific primer connects FAM or HEX fluorescent linker sequence.
Further, application of the molecular labeling in Rice Resistance characteristic of disease assistant breeding, includes the following steps:
S1, rice sample gene group DNA is extracted；
S2, using oryza sativa genomic dna as template, utilize above-mentioned primer combination carry out KASP reaction detection；Wherein, Liang Tiaote
Specific primer is separately connected different fluorescence sequences；
S3, selection detect that the rice sample of the corresponding fluorescence signal of the connected fluorescence sequence of primer PrimerY carries out breeding.
Beneficial effects of the present invention:
(1) SNP marker and Pigm gene close linkage that the present invention develops, by segregating population rice blast phenotypic evaluation
Afterwards, the SNP marker Phenotypic Selection efficiency for verifying exploitation reaches 100%, can in the different germ plasm resource such as long-grained nonglutinous rice, japonica rice quickly,
Accurately detect wide spectrum blast resistant gene Pigm.
(2) the SNP marker combination KASP genotyping technique that the present invention develops, can applying in quotient with high, medium and low flux
In industry chemoattractant molecule breeding, and the red tapes such as digestion, electrophoresis and sequencing are not needed in the detection process, reduce aerosol
The use of the Toxics such as pollution and EB, directly detects base, accuracy is not influenced by expanding fragment length, Er Qiejian
Just, quickly, accurately, high degree of automation substantially increases gene Breeding Efficiency, reduces costs.
(3) SNP marker of the present invention can carry out foreground selection and Foreground selection in seedling stage, reduce breeding population
Field planting scale, shorten the breeding time limit, accelerate breeding process.
Detailed description of the invention
Fig. 1 is the development process figure of this hair blast resistant gene Pigm molecular labeling.
Fig. 2 is to detect natural population's parting figure using molecular labeling K_060508.
Specific embodiment
The present invention will be described in detail below with reference to the accompanying drawings and embodiments.Experimental method used in embodiment is such as
It is conventional method without specified otherwise；Material, reagent used etc., are commercially available unless otherwise specified.
It is only to be not intended to limit the invention merely for for the purpose of describing particular embodiments in terminology used in the present invention.
It is also intended in the present invention and the "an" of singular used in the attached claims, " described " and "the" including majority
Form, unless the context clearly indicates other meaning.It is also understood that term "and/or" used herein refers to and wraps
It may be combined containing one or more associated any or all of project listed.
The preparation of 1 blast resistant gene Pigm molecular labeling of embodiment
1, design of primers
Pigm gene location is anchored on to the section 10387509-10390465 of No. 6 chromosomes of rice according to pertinent literature,
Respectively expand 50kb to two sides centered on gene interval, carries out SNP according to 3000 parts of rice weight sequencing datas of International Rice institute
Point extracts, the SNP site picked out, and extracts flanking sequence, and utilize online design of primers website BatchPrimer3
(http://probes.pw.usda.gov/batchprimer3/) carries out design of primers to it.Every group echo has three primers,
Two ends of specific primer 5 ' are separately connected FAM and HEX fluorescence sequence wherein.Primer entrusts the synthesis of Invitrogen company.
If sample P CR product only detects primer PrimerX and corresponds to fluorescence signal, detection site is bases G, is determined
The rice sample of test is free of Pigm gene；If only detecting primer PrimerY corresponds to fluorescence signal, detection site is base
A, the rice sample of discriminating test contain homozygous Pigm gene；Detection site is G if being detected simultaneously by two kinds of fluorescence signals:
A judges the Pigm gene that rice to be measured contains heterozygosis.
2, DNA is extracted: genomic DNA is extracted from rice leaf, using simplified CTAB method.
3, KASP reaction test
KASP reaction test carries out in LGC SNPline genotyping platform.20ng is added in micro reaction plate
DNA sample, is added KASP reaction mixture after drying, reaction system is shown in Table 2.PCR amplification is completed in water-bath thermal cycler,
Touchdown PCR reaction condition are as follows: 94 DEG C initial denaturation 15 minutes；First step amplified reaction, 94 DEG C be denaturalized 20 seconds, 65 DEG C~57
It DEG C anneals and extends 60 seconds, the temperature of 10 circulations, each cycle annealing and extension reduces by 0.8 DEG C；Second step amplified reaction, 94
It DEG C denaturation 20 seconds, anneals for 57 DEG C and simultaneously extends 60 seconds, 26 circulations.It is anti-to KASP using scanner Pherastar after the reaction was completed
Product is answered to carry out fluorescence data reading, the result of fluorescent scanning can be converted to figure automatically.
LGC SNPline genotyping platform used in the present invention and its matched reagent consumptive material are purchased from Britain LGC public affairs
Department.
The reaction system of 2 KASP of table detection
| Final concentration | Volume (μ L) | |
| 100UMPrimerC | 0.42μM | 0.0125 |
| 100UMPrimerY | 0.17μM | 0.0050 |
| 100UMPrimerX | 0.17μM | 0.0050 |
| 2xKASPMasterMix | 1x | 1.4792 |
| Ultrapure water | 1.4983 | |
| Total volume | 3 |
4, typing data is marked
K_060508 is marked to carry out Pigm genetic donor material paddy plum No. 4 and other 21 without Pigm rice varieties
The reaction verifying of KASP primary dcreening operation, the results are shown in Table 3.Pigm donor material paddy plum No. 4 are alkali in K_060508 test site testing result
Base A, 20 parts of rice varieties either other blast resistant gene donors or susceptible materials without Pigm are in test site
Detect bases G.It picks out and is isolated with Pigm donor material and SNP marker K_060508 that expanding effect is good.
Table 3 marks K_060508 primary dcreening operation typing data
| Material | Material explanation | Detection |
| 75-1- | Pi9 donor | G |
| C101 | Pi2 donor | G |
| Sixteen | Pi50 donor | G |
| Fukun | Pi-z donor | G |
| Paddy plum 4 | Pigm donor | A |
| Good fortune brocade | Pi-z+Pi-sh donor | G |
| BL6 | Pi1+Pi2 donor | G |
| Asom | Susceptible material | G |
| Cpslo | Susceptible material | G |
| Hunan is early | Susceptible material | G |
| It is former rich | Susceptible material | G |
| Lijing | Susceptible material | G |
| CO39 | Susceptible material | G |
| Hunan is short | Susceptible material | G |
| Kasal | Susceptible material | G |
| Close sun | Susceptible material | G |
| It is bright extensive | Susceptible material | G |
| Ⅱ | Susceptible material | G |
| Middle 9B | Susceptible material | G |
| Huang Hua | Susceptible material | G |
| Gold | Susceptible material | G |
| Japan | Susceptible material | G |
The group of 2 blast resistant gene Pigm molecular labeling of embodiment and label phenotype verifying
1, natural population verifies
For the specificity and practicability marked in the detection present invention, 187 parts of materials are examined using label K_060508
It surveys and verifies.Include the known kind containing homozygous Pigm gene in 187 parts of materials, contain the confession of other rice blast resistance genes
Body, general sense material, common hybridization rice and core rice breed.Mark genotyping result in natural population as shown in Fig. 2,
The kind of the gene containing Pigm known to 4 parts is detected as the homozygous Pigm genotype with rice blast resistance, detects in 2 parts of hybrid paddy rices
Heterozygosis Pigm genotype, for 6 parts of materials without amplified signal, remaining contains the donor of other blast resistant genes, general sense material, common
Hybrid paddy rice and core rice breed are detected as no rice blast resistance homozygosis pigm genotype.
As it can be seen that SNP marker K_060508 detection Pigm gene loci is the resistance locus of high specific, it can convenient and efficient
For identifying in rice varieties whether contain Pigm gene.
2, label phenotype verifying
It is carried out using the F2:3 family that label K_060508 constructs Pigm donor parents paddy plum No. 4 and receptor parent CO39
Detection.Meanwhile in rice seedling, rice blast bacterial strain CHL506 is inoculated with to 20 F2:3 familys and 2 parents, investigates the state of an illness, table
Type data fit like a glove with genotype, demonstrate feasibility and accuracy (being shown in Table 4) of the invention again.
The hereditary segregating population phenotype of table 4 and genotype identification
Although above the present invention is described in detail with a general description of the specific embodiments, with
Upper described is only presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the spirit and principles in the present invention it
Interior, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
Sequence table
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<120>development and application of the SNP marker of rice blast resistant gene Pigm
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Claims (10)
1. a kind of with blast resistant gene Pigm close linkage SNP marker, which is characterized in that the molecular labeling be with
The SNP marker K_060508 that blast resistant gene Pigm is isolated, the SNP marker detect the of No. 6 chromosome of rice
Base is G or A at 10421726 sites.
2. a kind of primer for detecting molecular labeling described in claim 1, which combines, includes:
(1) two specific primer: Primer X:5 '-CCAAGCAAATTACCACAACG-3 '；Primer Y:5 '-
CCAAGCAAATTACCACAACA-3'；
(2) universal primers: Primer C:5 '-TTGTTATACGGTTTAATTAAGGTGA-3 '.
3. the application in Markers for Detection blast resistant gene Pigm described in claim 1.
4. application of the molecular labeling described in claim 1 in blast resistant gene Pigm assistant breeding.
5. application of the molecular labeling described in claim 1 in the rice pest insects of breeding blast resisting.
6. the application in the rice pest insects of breeding blast resisting according to claim 5, which is characterized in that the application packet
It includes and carries out Pigm parting using KASP or Pigm is anchored in genetic chip.
7. application according to claim 3, which comprises the steps of:
S1, rice sample gene group DNA is extracted；
S2, using oryza sativa genomic dna as template, utilize primer described in claim 2 combination carry out KASP reaction detection；Wherein,
Two specific primers are separately connected different fluorescence sequences；
If S3, only detecting the corresponding fluorescence signal of the connected fluorescence sequence of primer Primer Y, detection site is base A, is sentenced
Rice sample of cutting off the water supply is the pure and mild type for carrying blast resistant gene Pigm；If only detecting the connected fluorescence sequence pair of primer Primer X
The fluorescence signal answered, then detection site is bases G, judges rice sample for the pure and mild type without blast resistant gene Pigm；If
Two kinds of fluorescence are detected simultaneously by, then judge rice sample for the heterozygous of blast resistant gene Pigm.
8. application according to claim 7, which is characterized in that primer Primer Y, Primer X 5 ' sections connection FAM or
HEX fluorescent linker sequence.
9. application of the molecular labeling described in claim 4 in Rice Resistance characteristic of disease assistant breeding, which is characterized in that including walking as follows
It is rapid:
S1, rice sample gene group DNA is extracted；
S2, using oryza sativa genomic dna as template, utilize primer described in claim 2 combination carry out KASP reaction detection；Wherein,
Two specific primers are separately connected different fluorescence sequences；
S3, selection detect that the rice sample of the corresponding fluorescence signal of the connected fluorescence sequence of primer Primer Y carries out breeding.
10. a kind of kit for the SNP marker for detecting blast resistant gene Pigm, the kit include such as claim
The combination of primer described in 2.
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Cited By (6)
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| CN110257553A (en) * | 2019-08-05 | 2019-09-20 | 江苏省农业科学院 | A kind of KASP molecule labelling method for identifying resistance gene of rice blast Pigm |
| CN110408719A (en) * | 2019-08-05 | 2019-11-05 | 江苏省农业科学院 | A kind of four primer molecule labeling methods for identifying rice blast resistant gene Pigm |
| CN112522432A (en) * | 2020-12-17 | 2021-03-19 | 华智生物技术有限公司 | Molecular marker for assisted breeding of rice blast resistance gene Bsr-d1 and application thereof |
| CN113046349A (en) * | 2020-12-25 | 2021-06-29 | 华智生物技术有限公司 | SNP molecular marker combination for detecting rice Wx gene and application thereof |
| CN114395641A (en) * | 2022-01-19 | 2022-04-26 | 山东省农业科学院 | Primer pair and kit for detecting blast disease-resistant gene PigmR, application of primer pair and kit and detection method |
| CN117402994A (en) * | 2023-10-18 | 2024-01-16 | 辽宁省水稻研究所 | SNP molecular marker of rice blast resistance gene Pi50, primer set and application |
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| CN110257553A (en) * | 2019-08-05 | 2019-09-20 | 江苏省农业科学院 | A kind of KASP molecule labelling method for identifying resistance gene of rice blast Pigm |
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| CN110408719B (en) * | 2019-08-05 | 2022-07-08 | 江苏省农业科学院 | Four-primer molecular marking method for identifying rice blast resistance gene Pigm |
| CN112522432A (en) * | 2020-12-17 | 2021-03-19 | 华智生物技术有限公司 | Molecular marker for assisted breeding of rice blast resistance gene Bsr-d1 and application thereof |
| CN113046349A (en) * | 2020-12-25 | 2021-06-29 | 华智生物技术有限公司 | SNP molecular marker combination for detecting rice Wx gene and application thereof |
| CN114395641A (en) * | 2022-01-19 | 2022-04-26 | 山东省农业科学院 | Primer pair and kit for detecting blast disease-resistant gene PigmR, application of primer pair and kit and detection method |
| CN117402994A (en) * | 2023-10-18 | 2024-01-16 | 辽宁省水稻研究所 | SNP molecular marker of rice blast resistance gene Pi50, primer set and application |
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