CN109913582A - A kind of molecular labeling and its application with resistance gene of rice blast close linkage - Google Patents
A kind of molecular labeling and its application with resistance gene of rice blast close linkage Download PDFInfo
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Abstract
A kind of molecular labeling and its application with resistance gene of rice blast close linkage, the molecular labeling primer include forward and reverse primer pair, and nucleotide sequence difference is as follows: InDel-19 is positive: 5 ' AAGGAGATCTGGTATGTGTGCG 3 ';InDel-19 is reversed: 5 ' AGTTTGGGGTAGTGAAATGCGA 3 ';InDel-25 is positive: 5 ' CCTGGTCTAAAGCGCACCTA 3 ';InDel-25 is reversed: 5 ' CGCCATGGATTCGTTCGACT 3 ';InDel-27 is positive: 5 ' TCGGTGCTTTAGATATGTTTTGCT 3 ';InDel-27 is reversed: 5 ' ACAACTCAAACCAAGCTTCTCA 3 '.It is used to detect the rice blast resistance gene contained, has very strong resistance, and resistance to rice blast, anti-spectrum is wide.
Description
Technical field
The present invention relates to Genetic and breeding in rice and molecular biology field, refer in particular to a kind of and Rice Resistance To Rice Blast base
Molecular labeling and its application because of close linkage.
Background technique
Rice is one of most important cereal crops in the whole world, brings up more than half population of the world.With rice routine
The yield level of the improvement of Breeding Strategies and constantly improve for molecular breeding technology, rice is also gradually increased, but rice pest
The lasting further development for hindering rice in breeding, wherein disease cause the loss of rice yield about more than
30% or more, it is especially showed with rice blast extremely serious.By fungiMagnaporthe oryzaeCaused rice blast be rice most
With one of disease dissolvingly, occur to cause sternly in the whole nation or even each rice region in the whole world, plantation and production to rice
The influence of weight.
Currently, the general strategy for preventing and treating rice blast is chemical prevention and cultivation resistant variety.Although chemical prevention can play
Certain effect, but the raising of peasant's grain cost is often resulted in, and drug effect can be greatly reduced after a number of uses, while can make
At the pollution of environment, have to the mankind potentially hazardous.Breeding and plantation resistant variety be prevention and treatment rice blast it is most economical, effectively and peace
Full measure, also complies with requirement of the mankind to green food, but simultaneously because rice blast pathogen Race frequently and
Different rice varieties have very strong specificity to different biological strains, and the disease-resistant variety for causing some single is in plantation 3-5
Disease-resistant characteristic is gradually lost after year, therefore is excavated and become breeding with resistance to rice blsat characteristic water using broad-spectrum resistance gene
The most economical effective method of rice varieties.Also have great importance in terms of ecological safety and environmental protection simultaneously.At present
Report isolated blast resisting major gene resistance a at least more than 80 from different rice varieties, wherein a gene more than 20 is
By successful clone, but the gene with resistance of wide spectrum is seldom.
On the other hand, traditional Rice Resistance To Rice Blast breeding is poor there are time-consuming, laborious, accuracy and vulnerable to environmental factor
The drawbacks of influence.Currently, with the development of molecular biology, the molecular labeling auxiliary based on Molecular Marker Assisted Selection Technology is educated
Kind, it, can be chain by directly detecting in laboratory using molecular labeling and the characteristics of blast resisting target gene close linkage
Molecular labeling, that is, can determine whether the presence and state of target gene, achieve the purpose that selection target character, and be in rice seedling
It is detectable, have the advantages that detection period is early, detection speed is fast, result is accurate and is not interfered by environmental condition.Therefore, it excavates
The new excellent genes with wide spectrum blast resisting characteristic, and develop therewith close linkage molecular labeling become further open
Open up the key of Rice Resistance To Rice Blast breeding.
Summary of the invention
The present invention provides a kind of and resistance gene of rice blast close linkage molecular labeling and its application, main mesh
Be develop a kind of molecular labeling, for overcoming the period in conventional rice rice blast resistance breeding method long, low efficiency, be not allowed
The defect really and vulnerable to bad border factor influenced.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
A kind of molecular labeling with resistance gene of rice blast close linkage, the molecular labeling primer include forward and reverse
Primer pair, nucleotide sequence difference are as follows:
InDel-19 is positive: 5 ' AAGGAGATCTGGTATGTGTGCG 3 ';
InDel-19 is reversed: 5 ' AGTTTGGGGTAGTGAAATGCGA 3 ';
InDel-25 is positive: 5 ' CCTGGTCTAAAGCGCACCTA 3 ';
InDel-25 is reversed: 5 ' CGCCATGGATTCGTTCGACT 3 ';
InDel-27 is positive: 5 ' TCGGTGCTTTAGATATGTTTTGCT 3 ';
InDel-27 is reversed: 5 ' ACAACTCAAACCAAGCTTCTCA 3 '.
Further, the Molecular mapping is located at Pi2/Pi9 multiple allele cluster on No. 6 chromosome of rice
In region.
Further, the kind of the rice are as follows: using with wide spectrum blast resisting characteristic kind Kang Feng B as male parent, with sense
Sick kind Lijiang xintuanheigu is female parent.
A kind of application with the molecular labeling of resistance gene of rice blast close linkage, is used for resistant rice cultivars offspring
Assisted selection.
Compared to the prior art, the beneficial effect that the present invention generates is:
1, the molecular labeling that present invention exploitation obtains has rice blast very strong for detecting the rice blast resistance gene contained
Resistance, and resistance, anti-spectrum is wide.
2, the resistance gene of rice blast in the present invention, navigated to is located at No. 6 chromosome Pi2/Pi9 allele
In cluster region and Pi2/Pi9 multiple allele equipotential or close linkage, therefore the molecular labeling that the present invention develops can conduct simultaneously
The close linkage label of these multiple equipotential disease-resistant genes, so as to can be used for Pi2/Pi9 gene in rice resistance breeding
The pyramiding breeding of molecular marker assisted selection or multiple resistant genes.
3, the present invention is by molecular marker assisted selection breeding, can greatly reduce breeding material in the planting scale in field and
The workload of later period identification effectively accelerates breeding process to significantly save human and material resources and financial resources.
Detailed description of the invention
Fig. 1 is SSR molecular marker and insertion and deletion InDel label and resistance gene of rice blast Pi-kf2 in the present invention
(t) genetic linkage maps and genetic distance schematic diagram between.
Fig. 2 is linkage molecule label and rice blast resistant gene Pi-kf2(t in the present invention) reality on chromosome
Physical location schematic diagram marks the corresponding linked marker of digital representation inside lower square brackets in the susceptible group's genotype of 220 F2
The exchange single plant number occurred in detection process.
Fig. 3 is that insertion and deletion marks InDel-19 to the genotype detection and linkage analysis of 45 susceptible single plants of F2;In figure,
1: disease-resistant parent Kang Feng B(KFB);2: Susceptible parent Lijiang xintuanheigu (LTH);3: disease-resistant gene pond (RP);4: susceptible gene
Pond (SP);The susceptible single plant of S:45 F2.
Fig. 4 is that insertion and deletion marks InDel-25 to the genotype detection and linkage analysis of 45 susceptible single plants of F2;Figure
In, 1: disease-resistant parent Kang Feng B(KFB);2: disease-resistant gene pond (RP);3: Susceptible parent Lijiang xintuanheigu (LTH);4: susceptible base
Because of pond (SP);The susceptible single plant of S:45 F2.
Fig. 5 is that insertion and deletion marks InDel-27 to the genotype detection and linkage analysis 1 of 45 susceptible single plants of F2: disease-resistant
Parent Kang Feng B(KFB);2: disease-resistant gene pond (RP);3: Susceptible parent Lijiang xintuanheigu (LTH);4: susceptible gene pool (SP);
The susceptible single plant of S:45 F2.
Fig. 6 be insertion and deletion mark InDel-19 to Kang Feng B/ OryzasativaLcv.Nipponbare hybridize F2 separate offspring 5 it is disease-resistant and 5 susceptible
The verifying of single plant;In figure, 1: disease-resistant parent Kang Feng B(KFB);2: Susceptible parent OryzasativaLcv.Nipponbare;R:F2 separates the disease-resistant single plant of offspring;S:
F2 separates the susceptible single plant of offspring.
Fig. 7 be insertion and deletion mark InDel-25 to Kang Feng B/ OryzasativaLcv.Nipponbare hybridize F2 separate offspring 5 it is disease-resistant and 5 susceptible
The verifying of single plant;In figure, 1: disease-resistant parent Kang Feng B(KFB);2: Susceptible parent OryzasativaLcv.Nipponbare;R:F2 separates the disease-resistant single plant of offspring;S:
F2 separates the susceptible single plant of offspring.
Fig. 8 be insertion and deletion mark InDel-27 to Kang Feng B/ OryzasativaLcv.Nipponbare hybridize F2 separate offspring 5 it is disease-resistant and 5 susceptible
The verifying of single plant;In figure, 1: disease-resistant parent Kang Feng B(KFB);2: Susceptible parent OryzasativaLcv.Nipponbare;R:F2 separates the disease-resistant single plant of offspring;S:
F2 separates the susceptible single plant of offspring.
Specific embodiment
Illustrate a specific embodiment of the invention with reference to the accompanying drawings.
Embodiment one
A kind of molecular labeling with resistance gene of rice blast close linkage, the molecular labeling be by with Rice Resistance To Rice Blast
The site the insertion and deletion InDel exploitation of gene close linkage is transformed.The molecular labeling primer is drawn comprising forward and reverse
Object pair, nucleotide sequence difference are as follows:
InDel-19 is positive: 5 ' AAGGAGATCTGGTATGTGTGCG 3 ';
InDel-19 is reversed: 5 ' AGTTTGGGGTAGTGAAATGCGA 3 ';
InDel-25 is positive: 5 ' CCTGGTCTAAAGCGCACCTA 3 ';
InDel-25 is reversed: 5 ' CGCCATGGATTCGTTCGACT 3 ';
InDel-27 is positive: 5 ' TCGGTGCTTTAGATATGTTTTGCT 3 ';
InDel-27 is reversed: 5 ' ACAACTCAAACCAAGCTTCTCA 3 '.
Its rice blast resistance gene contained has very strong resistance, and resistance to rice blast, and anti-spectrum is wide.This implementation
In example, the kind of rice are as follows: be female parent with Lijiang xintuanheigu using Kang Feng B as male parent.
Specifically, the Molecular mapping on No. 6 chromosome of rice, is located at Pi2/Pi9 multiple allele cluster region
Interior and Pi2/Pi9 multiple allele equipotential or close linkage, therefore the molecular labeling that the present invention develops can be multiple as these simultaneously
The close linkage of equipotential disease-resistant gene marks, so as to can be used for the molecule mark of Pi2/Pi9 gene in rice resistance breeding
Remember the pyramiding breeding of assisted Selection or multiple resistant genes.
Referring to Fig.1, Fig. 2, Fig. 3, Fig. 4 and Fig. 5.A kind of and resistance gene of rice blast close linkage molecular labeling
Preparation method, comprising the following steps:
(1) using the rice varieties with wide spectrum blast resisting characteristic as male parent, susceptible variety is female parent, and building one is used for rice
The F2 segregating population of seasonal febrile diseases resistant gene positioning;
In the present embodiment, protected with three system's infertility with special nucleus genetic background of the autonomous breeding of Sanming Institute of Agriculture Science
Holding is anti-rice blast rice kind Kang Feng B(KFB) it is male parent, it is that maternal progress is miscellaneous with susceptible variety Lijiang xintuanheigu (LTH)
It hands over and obtains F1, obtain F2 segregating population after F1 selfing.Rice blast pathogen is carried out in the F2 segregating population rice shoot 3-4 leaf phase manually to infuse
It penetrates and connects bacterium, be put into 24-26 DEG C of constant incubator dark culture for 24 hours after connecing bacterium, relative humidity 95-100% then moves into 25 DEG C of greenhouses
After interior culture 8-10 days, its incidence is observed and counts, identification obtains 220 susceptible single plants for connecting from group offspring
The analysis of lock relationship.
(2) the extremely disease-resistant single plant of at least ten and the extremely susceptible single plant of at least ten are randomly selected from F2 segregating population
Disease-resistant gene pond (RP) and susceptible gene pool (SP) are constructed respectively;
In the present embodiment, 15 extremely disease-resistant single plants and 15 extremely susceptible single plants are randomly selected from F2 segregating population, then use
The CTAB method of improvement extracts its leaves genomic DNA respectively, measures its concentration using DU800 spectrophotometric, and be uniformly diluted to
50ng/ μ L, by after dilution 15 disease-resistant single plant DNA and 15 susceptible single plant DNA difference mixed in equal amounts constitute disease-resistant gene ponds
Linkage analysis is used for susceptible gene pool.
(3) genome of two Parent Seedlings and two parent filial generation seedling is extracted respectively using the CTAB method of improvement
DNA;I.e. extract Kang Feng B(KFB), Lijiang xintuanheigu (LTH), F1 and F2 single-strain blade genomic DNA, the specific steps are as follows:
1) 0.1g rice leaf is weighed to be placed in grind into powder in liquid nitrogen and be transferred in 2.0ml centrifuge tube;
2) 800 μ L CTAB extracting solutions are added into the 2.0ml centrifuge tube, gently vibrates to mixing well, places water-bath
In 65 DEG C of warm bath 30min;
3) after taking-up is cooled to room temperature in water-bath, addition chloroform: isoamyl alcohol is the mixed liquor of 24:1 made from step 2
600 μ L, teetertotter and mix well, and obtain mixed liquor;
4) mixed liquor is centrifuged 10min with 12000rpm, then the supernatant of the mixed liquor is transferred to 1.5mL centrifuge tube
In;
5) isopropanol being pre-chilled that volume is 2 times of the supernatant is added into the 1.5mL centrifuge tube, shakes gently mixed
Even to visible DNA flocculent deposit generates;
6) the 1.5mL centrifuge tube is placed in centrifuge and 3min is centrifuged with 5000rpm, abandon supernatant;
7) 75% 500 μ L of ethyl alcohol cleaning is added toward the 1.5mL centrifuge tube, then 3min is centrifuged with 5000rpm, abandons supernatant;
8) step 7) is repeated, the DNA washed is obtained;
9) the DNA placement super-clean bench washed is dried up and is dissolved in 200 μ L high pressure ddH2It is standby to place 4 DEG C of refrigerators preservations by O
With.
(4) according to rice biological informatics database (http://archive.gramene.org/markers/
Microsat the SSR molecular marker) published, by polymorphism between two parents of screening and two gene pools, acquisition and rice blast
The chain molecular labeling of resistant gene, to carry out Primary Location to rice blast resistance gene;
Specifically, further being carried out to the susceptible group of F2 in step (1) in conjunction with linkage analysis software Mapmaker/EXP 3.0
Genotype identification and linkage analysis, and Relative Hereditary distance, acquisition and mesh are converted for recombination fraction by Kosambi operation function
Molecular labeling Rm19776, Rm19781, Rm527, Rm7213, Rm5850 and the Rm7311 for marking gene linkage, by rice blast resistance
Gene carries out Primary Location between Rm527 and Rm7213, these labels go out altogether during to 220 F2 susceptible crowd surveillance
45 exchange recombination single plants are showed and target gene have certain genetic distance;
(5) japonica rice and two subspecies of the long-grained nonglutinous rice dyeing of Primary Location in step (4) that genome sequencing is completed are compared
Genome sequence polymorphism in body region, then finely positioning is carried out to rice blast resistance related gene, to obtain and resist
The site insertion and deletion InDel of rice blast ospc gene close linkage, then the site insertion and deletion InDel is utilized into Primer
5.0 software design of Premier is simultaneously converted into relative specific insertion and deletion InDel molecular labeling.
In the present embodiment, in order to develop related molecular marker for target gene further progress finely positioning, japonica rice is compared
9311 liang of subspecies of OryzasativaLcv.Nipponbare and long-grained nonglutinous rice genome sequence polymorphism in the chromosomal region of Primary Location in step (4),
The site insertion and deletion InDel with blast resistant gene close linkage is obtained, 5.0 software design of Primer Premier is utilized
And it is translated into corresponding specificity InDel molecular labeling, obtain the label with target blast resistant gene close linkage
InDel-19, InDel-22, InDel-25 and InDel-27, in group's genotyping susceptible to F2, discovery is in addition to InDel-
22 labels are outer, other three labels do not occur exchange recombination single plant and target gene isolates, and there are close linkage relationships.
The applicant has by bulk segregant analysis method and recessive population analysis method, and using molecular marking technique from one
Have special nucleus genetic background Three-line rice infertility holding be kind Kang Feng B(KFB) in separation, identified a wide spectrum rice blast
Sick resistant gene, and by exploitation close linkage molecular labeling by its finely positioning on No. 6 chromosome of rice, be located at
In Pi2/Pi9 allele cluster region.Pass through allele-specific molecular Marker Identification and the amplification sequencing of series of genes group
And sequence alignment analysis, it is found that rice blast resistance gene is different from other reported Pi2/Pi9 allele in Kang Feng B, pushes away
It surveys as a new rice blast resistance gene with resistance of wide spectrum.The exploitation and base that the present invention is marked by compact linkage molecule
The finely positioning of cause is that the further clone of the later period rice blast resistance gene and molecular mechanism research are laid a good foundation.
Referring to Fig.1, Fig. 2, Fig. 3, Fig. 4 and Fig. 5.A kind of and resistance gene of rice blast close linkage molecular labeling
Using the identification for resistance gene of rice blast and the assisted selection for resistant rice cultivars offspring.
Wherein, the method for the identification of resistance gene of rice blast includes the following steps:
A) using the DNA of rice single plant material to be identified as template;
B) PCR amplification is carried out to the DNA in step a) with molecular labeling and obtains amplified production;It is specifically applied in the present embodiment,
To carry out PCR amplification with molecular labeling InDel-19, InDel-25 and InDel-27;The reaction system of PCR amplification in this step 1
Based on 10 μ L of total volume, including following each component: 5.0 μ L PCR MasterMix, 0.5 μ L forward primer, 0.5 μ L reversely draw
Object, 0.8 μ L DNA, 3.2 μ L ddH2O;The reaction condition of PCR amplification are as follows: 94.0 DEG C of initial denaturation 5min;94.0 DEG C of denaturation 30S,
55 DEG C of renaturation 30S, 72 DEG C of extension 1min, 35 recycle;72 DEG C of extension 7min, 4 DEG C of preservations;
C) gel electrophoresis separation analysis is carried out to the amplified production in step b) using 8% non-denaturing polyacrylamide gel, led to
Nucleic acid staining dye observation is crossed, the banding pattern of different molecular weight size occurs;In this step, 8% non-denaturing polyacrylamide gel
Configuration method: based on 30 μ L of total volume, including following each component: 23.4mL ddH2O, 40% acrylamide of 6mL, 0.3mL 50
×CTAB,0.3mL APS,24.9μL TEMED;
If d) there is specific band corresponding with the molecular labeling in banding pattern, which is to contain rice blast
The plant of sick resistant gene, is specifically applied in the present embodiment, for that can amplify 241bp(InDel-19 respectively), 250bp
(InDel-25) and 243bp(InDel-27) single plant of specific band is the plant containing target resistant gene.
The present invention keeps system from a Three-line rice infertility with special nucleus genetic background by the method for map based cloning
Rice varieties Kang Feng B(KFB) in obtain the new rice blast resistance gene with resistance of wide spectrum, and be located at rice
On No. 6 chromosome, it is located in Pi2/Pi9 allele region.And the side compared using genome sequence between indica and japonica subspecies
Method is developed to obtain the insertion and deletion InDel molecular labeling of 3 He target resistant gene close linkage, anti-in later period rice blast
Property breeding in, can by the molecular labeling of these close linkages carry out assisted Selection, can not only overcome in conventional breeding methods
Period is long, low efficiency, inaccuracy and the disadvantages of influence vulnerable to bad border factor, while carrying out in laboratory with can having Objective
The identification of disease-resistant gene, and purposefully carry out the polymerization of multiple disease-resistant genes, thus cultivate with resistance of wide spectrum and
The new rice variety of lasting stability resistance.Meanwhile the exploitation of these compact linkage molecules label and the finely positioning of gene are to this
The further clone of resistant gene and molecular mechanism research are of great significance.
Embodiment two
Referring to Fig. 6, Fig. 7, Fig. 8.In order to further prove molecule that the present invention develops and rice blast resistance gene close linkage
The application in rice resistance assisted selection is marked, the applicant is with disease-resistant variety Kang Feng B(KFB) it is male parent, with susceptible
Kind OryzasativaLcv.Nipponbare is that female parent is configured with a new cross combination, and obtains a new F2 segregating population, passes through embodiment
Bacterium is connect to new F2 segregating population in the method for middle step (1), obtains the disease-resistant single plant of a certain number of F2 and susceptible single plant.
Referring concurrently to the identification method of resistance gene of rice blast in embodiment one, these three insertion and deletions InDel label point is utilized
It is other that polymorphism analysis is carried out to two parents, it is found that two parents have polymorphism in these three sites InDel, shows as different points
The amplification banding pattern of son amount size, further detects the susceptible single plant of the disease-resistant single plant in the part F2 and part, finds disease-resistant single plant
Banding pattern it is consistent with disease-resistant parent Kang Feng B(KFB), the banding pattern of susceptible single plant is consistent with Susceptible parent OryzasativaLcv.Nipponbare, Fig. 6,7,8 distinguish
Be molecular labeling InDe-19, InDel-25 and InDel-27 to OryzasativaLcv.Nipponbare/Kang Feng B(KFB) hybridization the disease-resistant list of F2 population segment
The detection of strain and the susceptible single plant in part.Illustrate insertion and deletion that the present invention develops and rice blast resistance gene close linkage
InDel label can accurately distinguish disease-resistant and susceptible single plant, therefore can be applied to later period rice anti-rice blast molecule well
Marker assisted selection breeding.Using these labels to rice seedling genome carry out PCR amplification can accurate judgement rice blast it is anti-
Property the presence or absence of gene and state, rice resistance breeding selection efficiency can be significantly improved, reduce the work of later period Field Screening identification
It measures, accelerates breeding process.
The above is only a specific embodiment of the present invention, but the design concept of the present invention is not limited to this, all to utilize this
Design makes a non-material change to the present invention, and should all belong to behavior that violates the scope of protection of the present invention.
Claims (4)
1. a kind of and resistance gene of rice blast close linkage molecular labeling, it is characterised in that: the molecular labeling primer
Comprising forward and reverse primer pair, nucleotide sequence difference is as follows:
InDel-19 is positive: 5 ' AAGGAGATCTGGTATGTGTGCG 3 ';
InDel-19 is reversed: 5 ' AGTTTGGGGTAGTGAAATGCGA 3 ';
InDel-25 is positive: 5 ' CCTGGTCTAAAGCGCACCTA 3 ';
InDel-25 is reversed: 5 ' CGCCATGGATTCGTTCGACT 3 ';
InDel-27 is positive: 5 ' TCGGTGCTTTAGATATGTTTTGCT 3 ';
InDel-27 is reversed: 5 ' ACAACTCAAACCAAGCTTCTCA 3 '.
2. a kind of molecular labeling with resistance gene of rice blast close linkage as described in claim 1, it is characterised in that: institute
Molecular mapping is stated on No. 6 chromosome of rice, is located in Pi2/Pi9 multiple allele cluster region.
3. a kind of molecular labeling with resistance gene of rice blast close linkage as described in claim 1, it is characterised in that: institute
State the kind of rice are as follows:, as male parent, to be with susceptible variety Lijiang xintuanheigu with wide spectrum blast resisting characteristic kind Kang Feng B
It is maternal.
4. a kind of application with the molecular labeling of resistance gene of rice blast close linkage, it is characterised in that: be used for Rice Resistance
The assisted selection of sick kind offspring.
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Cited By (5)
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CN111187851A (en) * | 2019-10-08 | 2020-05-22 | 三明市农业科学研究院 | Specific molecular marker primer of rice blast resistance gene Pi-kf2(t) and application |
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CN112680537A (en) * | 2020-12-21 | 2021-04-20 | 云南省农业科学院农业环境资源研究所 | Coseparation molecular marker of rice broad-spectrum rice blast resistance gene Pi57 and application thereof |
CN112680537B (en) * | 2020-12-21 | 2021-10-08 | 云南省农业科学院农业环境资源研究所 | Coseparation molecular marker of rice broad-spectrum rice blast resistance gene Pi57 and application thereof |
CN115094158A (en) * | 2021-11-12 | 2022-09-23 | 江苏省农业科学院宿迁农科所 | KASP marker development of rice blast resistance gene Pid4 and application thereof |
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