CN109628629A - The SNP marker development and application of rice bacterial leaf spot resistant gene xa5 - Google Patents
The SNP marker development and application of rice bacterial leaf spot resistant gene xa5 Download PDFInfo
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Abstract
The present invention provides a kind of and bacterial leaf spot resistant gene xa5 close linkage SNP marker K_050527, it is C or G that the SNP marker, which detects base at the 437500th site of No. 5 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 bacterial leaf spot resistant gene xa5 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 bacterial leaf spot resistant gene xa5；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 ethidium bromide 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 a kind of rice bacterial leaf spot resistant gene xa5's
SNP marker development and application.
Background technique
Bacterial blight of rice is one of three major disease of rice, by Xanthomonas campestris (Xoo, Xanthomonas oryzae
Pv.Oryzae) cause, be most important paddy bacterial disease in the world, rice region morbidity is heavier in south China, can cause the underproduction
20%-50% can cause to have no harvest when serious.However, still lacking the chemistry for effectively preventing bacterial blight of rice in current production
Pesticide, so cultivating and plant disease-resistant variety is the prevention and treatment most economical effective measures of bacterial leaf-blight.In recent decades, with molecule
The development of science of heredity, molecular marker assisted selection play very important effect in paddy disease-resistant breeding, using efficient
The single plant that molecular marker assisted selection contains target gene is hybridized, and can precisely carry out the breeding of objective trait, and can subtract
Small breeding population field planting scale shortens the breeding time limit, saves breeding cost.
So far, there is a bacterial leaf spot resistance gene more than 40 identified and clone.Xa1,Xa3,Xa26,xa5,
Xa10, xa13, Xa21, Xa23, xa25, Xa27, xa41 etc. 10 be by the main effect disease-resistant gene of successful clone, wherein xa5,
Xa13, xa25 and xa41 etc. 4 are recessive disease-resistant gene (Liu Guifu etc., 2018).
Xa5 is the recessive wide spectrum Bacterial blight resistance gene with important Breeding value, is located on No. 5 chromosome of rice,
CDNA overall length 906bp includes 3 exons, encodes a protein product being made of 106 amino acid, which is
The γ subunit (II A γ of TF) of one II A of transcription factor, II A γ of TF is Eukaryotic transcription factor, and gene structure is different from
Other typical NBS-LRR class disease-resistant genes.Xa5 gene has in disease-resistant variety IRBB5 and susceptible variety OryzasativaLcv.Nipponbare and IR24
The difference of two bases causes the in IRBB5 the 39th valine to become glutamic acid in OryzasativaLcv.Nipponbare and IR24, the amino acid
The difference in site causes the difference of disease resistance, and there is also illustrate xa5 genotype and disease-resistant table in other disease-resistant and susceptible varieties
The correlation of type is conservative (national rice data center).
The biological function of xa5 is analyzed, the results showed that the recessive disease resistance response that xa5 is mediated inhibits pathogen
Transfer rather than the proliferation (Iyer-Pascuzzi et al., 2008) for limiting pathogen.In addition, have the result shows that xa5 not only
Bacterial blight-resisting, while may also resistant (Xie et al., 2014) to bacterial stripe.Currently, detection xa5 gene
Label used is mostly RFLP and CAPS label, and with xa5 there are certain physical distance, testing result easily causes to judge by accident, RFLP
And CAPS label is only used for the polymorphism analysis between specific parental combination, experimental implementation is cumbersome, and detection flux is small, is not suitable for big
The germplasm identification or Resistance resource of scale screen.
For above situation, the functional SNP marker that exploitation is isolated with xa5 gene, using KASP technology to being developed
SNP marker carry out Genotyping, thus establish efficiently, environmental protection detection architecture, to promote xa5 gene in Hybrid breeding in commercial system
Large-scale application be of great significance.
Summary of the invention
The invention discloses the SNP marker development and application of rice bacterial leaf spot resistant gene xa5 a kind of, the molecular labeling is
The functional SNP marker isolated with wide spectrum bacterial leaf spot resistant gene xa5.The label is based on KASP technological development, high-throughput can examine
Survey the 437500th bit base of No. 5 chromosome of rice genome.Present invention application KASP technology carries out base to SNP marker
Because of parting, has many advantages, such as that easy to operate, low in cost, detection cycle is short, label is stable, can precisely detect bacterial blight of rice
Recessive disease-resistant gene xa5 is of great significance to the bacterial leaf spot resistance of improvement rice strain.
Specifically, a kind of development process of the molecular labeling of bacterial leaf spot resistance gene Xa5 of the present invention is shown in Fig. 1.
According to the literature, xa5 gene is led there are two the difference of base in disease-resistant variety IRBB5 and susceptible variety OryzasativaLcv.Nipponbare and IR24
It causes the 39th valine in IRBB5 to become glutamic acid in OryzasativaLcv.Nipponbare and IR24, and causes the difference of bacterial leaf spot resistance
It is different.One of them is selected, i.e., positioned at 437500 bit bases of No. 5 chromosome as candidate SNP locus.
Candidate SNP locus flanking sequence is extracted, and it is drawn using online design of primers website BatchPrimer3
Object design.Using label K_050527 first the rice donor kind to 6 parts containing xa5 gene and 11 parts of non-donor rice varieties into
The verifying of row KASP reaction test, then by natural population's popularization to 188 parts, expanding effect is good, and can accurately distinguish test
Material.In addition, being detected using the label to xa5 heredity segregating population, in conjunction with phenotypic data, it was confirmed that the label it is accurate
With it is efficient.
In order to solve the problems in the existing technology, the object of the present invention is to provide with bacterial leaf spot resistance gene Xa5
SNP (single nucleotide polymorphism) the molecular labeling K_050527 of close linkage and its application.
In order to achieve the object of the present invention, technical scheme is as follows:
A kind of SNP marker with bacterial leaf spot resistant gene xa5 close linkage, the molecular labeling are and bacterial leaf spot resistant base
Because of the SNP marker K_050527 that xa5 is isolated, which detects base at the 437500th site of No. 5 chromosome of rice
For C or G；
It is a kind of for detect molecular labeling described in claim 1 primer combine include: (1) two specific primer:
Primer X:5 '-TAAAGTAGATACCTTATCAAACTGG-3 '；Primer
Y5’-TAAAGTAGATACCTTATCAAACT
GC--3'；(2) universal primers: Primer C:5 '-CTCGACGAGATGGTCTCC-3 ' is specifically shown in Table 1.
1 flag sequence table of table
A kind of kit for the SNP marker detecting bacterial leaf spot resistant gene xa5, the kit includes above-mentioned primer
Combination.
The molecular labeling detection bacterial leaf spot resistant gene xa5, in bacterial leaf spot resistant gene xa5 assistant breeding and in breeding
Application in the rice pest insects of bacterial blight-resisting.
Further, the application carries out Xa5 parting including the use of KASP or Xa5 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 Primer Y, detection site is base
G judges rice sample for the pure and mild type of carrying bacterial leaf spot resistant gene xa5；If only detecting the connected fluorescence sequence of primer Primer X
Corresponding fluorescence signal is arranged, then detection site is base C, judges rice sample for the pure and mild type without bacterial leaf spot resistant gene xa5；
If being detected simultaneously by two kinds of fluorescence, judge rice sample for the heterozygous of bacterial leaf spot resistant gene xa5.
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 Xa5 gene close linkage that the present invention develops, by segregating population bacterial leaf-blight 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 bacterial leaf spot resistant gene xa5.
(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 bacterial leaf spot resistant gene xa5 molecular labeling.
Fig. 2 is to detect natural population's parting figure using molecular labeling K_050527.
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 bacterial leaf spot resistant gene xa5 molecular labeling of embodiment
1, design of primers
Xa5 gene order will have been delivered and OryzasativaLcv.Nipponbare reference gene group (MSU7.0) compares, determined the SNP at rice 5
437500 of chromosome.SNP site in sequence verification donor material extracts two sides 50bp centered on candidate SNP locus
Flanking sequence, and utilize online design of primers website BatchPrimer3 (http://probes.pw.usda.gov/
Batchprimer3/ design of primers) is carried out to it.Three primers are marked with, two for the design of critical sites base difference
Specific primer, a universal primer, two ends of specific primer 5 ' are separately connected FAM and HEX fluorescent linker sequence wherein.
Primer entrusts the synthesis of Invitrogen company.
If sample P CR product only detects primer PrimerY and corresponds to fluorescence signal, detection site is bases G, is determined
The rice sample of test is containing homozygous Xa5 gene；If only detecting primer Primer X corresponds to fluorescence signal, detection site is
Base C, the rice sample of discriminating test is without containing homozygous Xa5 gene；The check bit if being detected simultaneously by two kinds of fluorescence signals
Point is G:C, judges the Xa5 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.20~50ng 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) | |
| 100UM Primer C | 0.42μM | 0.0125 |
| 100UM Primer Y | 0.17μM | 0.0050 |
| 100UM Primer X | 0.17μM | 0.0050 |
| 2x KASP Master Mix | 1x | 1.4792 |
| Ultrapure water | 1.4983 | |
| Total volume | 3 |
4, typing data is marked
Rice varieties BOTESWAR and SAITA containing xa5 gene etc. are tested for 6 parts totally by sequencing with label K_050527
The genetic donor material of card and other 11 parts of rice varieties without xa5 gene carry out the reaction verifying of KASP primary dcreening operation, the results are shown in Table
3.The rice varieties of the gene containing xa5 are all detected as G type base in labeled test site, and 11 parts of non-donor rice varieties are being tested
Site primer goes out base C.
Table 3 marks K_050527 test cdna typing data
| Title material | Material explanation | K_050527 |
| BOTESWAR 2 | Xa5 donor | G |
| SAITA | Xa5 donor | G |
| UCP122 | Xa5 donor | G |
| Aus295 | Xa5 donor | G |
| Karia | Xa5 donor | G |
| BAZAIL 980 | Xa5 donor | G |
| V20B | C | |
| Bright extensive 86 | C | |
| IR24 | C | |
| Dular | C | |
| IR36 | C | |
| Balilla | C | |
| TP309 | C | |
| R900 | C | |
| In spend 11 | C | |
| 9311 | C | |
| NIP | C |
The group of 2 bacterial leaf spot resistant gene xa5 molecular labeling of embodiment and label phenotype verifying
1, natural population verifies
In order to detect the specificity and practicability of SNP marker K_050527, using label K_050527 to 188 parts of materials into
Row detection (see Fig. 2).It include the known kind containing xa5 gene, the confession containing other bacterial leaf spot resistant genes in 188 parts of materials
Body, general sense material, common hybridization rice and core rice breed.Mark the genotyping result such as following figure institute in natural population
Show, the kind containing xa5 gene known to 3 parts is detected as homozygous xa5 genotype, the donor containing other bacterial leaf spot resistant genes, general
Sense material and core rice breed are detected as no bacterial leaf spot resistance homozygosis Xa5 genotype other than the sample of no amplification.
As it can be seen that SNP marker K_050527 detection xa5 gene loci is the resistance locus of high specific, it can convenient and efficient
For identifying in rice varieties whether contain xa5 gene.
2, label phenotype verifying
Using label K_050527 to the kind UCP122 containing the xa5 gene and susceptible variety IR24 for being free of xa5 gene
The F2 heredity segregating population of building is detected.Meanwhile in rice booting latter stage, 40 F2 single plants and 2 parent materials are connect
The kind non-affine bacterial leaf spot bacterial strain FuJ of xa5 gene, carries out Disease investigation, homozygous G:G genotype all shows to resist after three weeks
Sick phenotype, phenotype 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
<110>Hua Zhi rice biological Technology Co., Ltd.
<120>development and application of the SNP marker of rice bacterial leaf spot resistant gene xa5
<160> 3
<170> Patent In version 3.3
<210> 1
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
taaagtagat accttatcaa actgg 25
<210> 2
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
taaagtagat accttatcaa actgc 25
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ctcgacgaga tggtctcc 18
Claims (10)
1. a kind of with bacterial leaf spot resistant gene xa5 close linkage SNP marker, which is characterized in that the molecular labeling be with
The SNP marker K_050527 that bacterial leaf spot resistant gene xa5 is isolated, the SNP marker detect the 437500th of No. 5 chromosome of rice
Base is C or G at site.
2. a kind of primer for detecting molecular labeling described in claim 1, which combines, includes:
(1) two specific primer: Primer X:5 '-TAAAGTAGATACCTTATCAAACTGG-3 '；Primer Y:5 '-
TAAAGTAGATACCTTATCAAACTGC-3'；
(2) universal primers: Primer C:5 '-CTCGACGAGATGGTCTCC-3 '.
3. application of the molecular labeling described in claim 1 in detection bacterial leaf spot resistant gene xa5.
4. application of the molecular labeling described in claim 1 in bacterial leaf spot resistant gene xa5 assistant breeding.
5. application of the molecular labeling described in claim 1 in the rice pest insects of breeding bacterial blight-resisting.
6. the application in the rice pest insects of breeding bacterial blight-resisting according to claim 5, which is characterized in that the application
Xa5 parting is carried out including the use of KASP or Xa5 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 bases G, is sentenced
Rice sample of cutting off the water supply is the pure and mild type for carrying bacterial leaf spot resistant gene xa5；If only detecting the connected fluorescence sequence pair of primer Primer X
The fluorescence signal answered, then detection site is base C, judges rice sample for the pure and mild type without bacterial leaf spot resistant gene xa5；If same
When detect two kinds of fluorescence, then judge rice sample for the heterozygous of bacterial leaf spot resistant gene xa5.
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 5 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 claim 2 primer combination carry out KASP reaction detection；Wherein, two
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 Primer Y carries out breeding.
10. a kind of kit for the SNP marker for detecting bacterial leaf spot resistant gene xa5, the kit include such as claim 2
Primer combination.
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| 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 |
| CN114480705A (en) * | 2022-01-13 | 2022-05-13 | 宁波市农业科学研究院 | SNP molecular marker of rice bacterial blight resistant gene XA23 and amplification primer and application thereof |
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| CN111926100A (en) * | 2020-08-25 | 2020-11-13 | 海南波莲水稻基因科技有限公司 | Molecular marker of rice bacterial leaf blight resistant gene xa5 and application thereof |
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| 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 |
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