CN106755475A - A kind of method of early 39 rice blast resistance genes in identification super early rice - Google Patents
A kind of method of early 39 rice blast resistance genes in identification super early rice Download PDFInfo
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Abstract
The invention discloses a kind of method for identifying early 39 rice blast resistance genes in super early rice, comprise the following steps：Rice blast inoculated identification；Extract the DNA of paddy rice sample to be detected；DNA to being extracted enters performing PCR amplification；Pcr amplification product is sequenced, the paddy rice sample DNA sequence containing pid2 allele is prepared；Pid2 allele fragments are converted into amino acid sequence, by the amino acid sequence of pid2 allele fragments and reference amino acid sequence (PID2：ACR15163.1) it is compared；If the amino acid series of pid2 allele have a nonsynonymous mutation I441M, i.e. its 441st amino acid by the methionine that isoleucine mutation is susceptible albumen, then the paddy rice sample to be detected has the susceptible allele of Pid2.Result shows that middle morning 39 includes that the material to be tested in its pedigree contains the disease-resistant allele of Pid2.
Description
Technical field
The invention belongs to genetic engineering field, specifically, it is related to early 39 rice blast resistances in a kind of identification super early rice
The method of gene.
Background technology
Paddy rice is the first generalized grain crop.In recent years, with social development, the further adjustment of agricultural planting structure, I
The grain security secret worry of state is protruded.Zao Xian rice because its shelf-stable, widely used, commodity is good the features such as, especially as
The effect of grain for industrial uses is greatly increased, and is the important support of China's grain security.Early 39 because of it in conventional super Indica rice kind
With high yield and high quality and the characteristics of high resistant to rice blast, super hybridization rice Demonstration And Extension kind was recommended as by the Ministry of Agriculture in continuous 6 years, it is several recently
The control material that year is tried as Zhejiang Province's morning rice region always.Early 39 even create two Zhejiang Agriculture rice high yields Ji Buddhist nuns in 2013
This record, as the kind that Zhejiang Province's early rice cultivated area is maximum.Middle morning 39 is widely used in early rice breeding as backbone parent
In, " excellent 39 " of strain two also passed through national authorization to the progeny material prepared by middle morning 39 in 2014.Middle morning 39 is used as super morning
Rice varieties not only show long-term high yield, and moderate growth duration is suitable to various cultivation modes, and high resistant to rice blast.In 2008-
In Zhejiang Province's early rice regional testings in 2009, the rice blast resistance of middle morning 39 is anti-or high anti-.Middle morning 39 was used as Zhejiang in recent years
Its anti-pest qualification result of early rice regional testing check variety is saved to resist or anti-for high.We are using from the whole nation 12 in early stage invention
146 bacterial strain centerings of individual provinces and cities early 39 have carried out the anti-spectrum of rice blast and have determined, and as a result show the average of 39 pairs of indica type microspecies of middle morning
Anti- spectrum is 82.9%, and the average anti-spectrum to round-grained rice type microspecies is 71.2%.39 pairs of anti-spectrums of the rice blast bacterial strain of difference provinces and cities of middle morning
It is inconsistent, the anti-spectrum of bacterial strain to Zhejiang, Guizhou and Guangxi is respectively 92.9%, 93.1% and 100%, and morning 39 exists in explanation
This 3 provinces can use as the anti-source of rice blast resistance breeding, for middle morning 39 region promote provide theoretical reference according to
According to.
Disease-resistant gene is the basis of resistance breeding.Identification, positioning and clone's wide spectrum blast resistant gene, water is understood to deep
The molecular mechanism of rice broad-spectrum disease resistance, cultivates wide spectrum and permanent disease-resistant kind, and continuous and effective control rice blast is significant.Mesh
Preceding Rice Blast resistance improvement, the main method taken is will to be returned after after improved materials with the hybridization of disease-resistant material, and
Disease-resistant offspring is screened by systematic breeding and Resistance Identification.However, resistant gene and these resistances to containing in disease-resistant material
The scarcity of the understanding such as the upper popular resistance against diseases of bacterial strain of gene pairs production, causing the selection of disease-resistant parent has stronger blindness
Property, this further have impact on the efficiency and effect of breeding for disease resistance.Although middle morning 39 since the authorization, has shown in extension process
Stronger rice blast resistance, however, with reference to " Gene-for-gene hypothesis " and field breeding practice, new pathogenic strong rice blast
Popular biological strain overcomes the risk of the rice blast resistance of middle morning 39 still to exist, and the extension of time is promoted with middle morning 39,
This risk more and more higher.Therefore, early 39 in identification in rice blast disease-resistant gene be conducive to clearly in early 39 rice blast resist
Property basis, be further resistance breeding using providing theoretical foundation.
The content of the invention
In view of this, the present invention is directed to above-mentioned problem, there is provided early 39 rice blast resistances in one kind identification super early rice
The method of gene.
In order to solve the above-mentioned technical problem, the invention discloses a kind of side for identifying early 39 rice blast resistances in super early rice
Method, comprises the steps：
1) with the rice varieties in the rice blast inoculation ground paddy B, middle morning 39 and its pedigree that infect middle morning 39；
2) DNA of paddy rice sample to be detected is extracted；
3) DNA extracted using pid2 primer pairs enters performing PCR amplification；
4) pcr amplification product is sequenced, and the DNA sequence dna after sequencing is manually proofreaded and hand through BioEdit softwares
Work is changed, and completes sequence assembly, prepares the paddy rice sample DNA sequence containing pid2 allele；By pid2 equipotential bases
Because fragment is converted to amino acid sequence；
5) result interpretation：
By the amino acid sequence of pid2 allele fragments and reference amino acid sequence (PID2：ACR15163.1) compared
Compared with；
If the amino acid series of pid2 allele have a nonsynonymous mutation I441M, i.e. its 441st amino acid by
Isoleucine mutation is the methionine of susceptible albumen, then the paddy rice sample to be detected is super with susceptible pid2 allele
Early 39 kinds in level early rice.
Further, pid2 primers include pid2 F primer and nucleotides of the nucleotide sequence as shown in SEQ ID No.1
Pid2 R primer of the sequence as shown in SEQ ID No.2.
Compared with prior art, the present invention can be obtained including following technique effect：
1) method of this research and utilization blast resistance identification, rice blast resistance gene Markers for Detection and sequencing, point
The hereditary basis of the rice blast durable resistance of middle morning 39 is analysed.Result shows：Middle morning 39, gold early 47 and middle group No. 3 to Zhejiang Province and
In Jiangxi Province dominate biological strain show water resistant high put down；Middle morning 39, gold early 47 and middle group No. 3 are to used in research 7
Rice blast resistance gene molecular labeling is positive band；Middle morning 39 is identical with the anti-spectrum of golden early 47, middle group No. 3.Middle morning
39 and gold early 47, middle group No. 3 either phenotype or genotype it is all completely the same, in explanation early 39 couples of biological strain 14-754 and
No. 3 are organized in the disease-resistant performance warp of rice blast of 16-754, from golden early 47.
2) the molecular sequences measurement result of rice blast resistance gene Pid2 shows that the material to be tested in the pedigree of middle morning 39 contains
There is the disease-resistant allele of Pid2.
Certainly, implement any product of the invention to it is not absolutely required to while reaching all the above technique effect.
Brief description of the drawings
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes a part of the invention, this hair
Bright schematic description and description does not constitute inappropriate limitation of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 is early 39 family trees in super early rice of the present invention；
Fig. 2 is rice blast resistance gene specific molecular marker detection of the present invention；Wherein, 1-9 is represented respectively：Zhejiang spoke 802,
It is good educate short No. 3 293, Hunan morning Xian 1, blue or green paddy, the early blue or green No. 1, gold early 47 in land, middle group No. 3, praise and educate 253 and middle morning 39；
Fig. 3 is amino acid alignment analysis of the present invention.
Specific embodiment
Describe embodiments of the present invention in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section can fully understand and implement according to this to solve technical problem and reach the implementation process of technology effect.
1 materials and methods
1.1 experiment materials
Middle morning 39 (educates 253 selection cross (China rice data center.http by middle group No. 3 with good://
www.ricedata.cn/variety/varis).The field resistance that its pedigree is reviewed with reference to each kind is showed, it is presumed that in
The resistance of early 39 pairs of rice blast be via middle group No. 3 from gold early 47, but be theoretically unsound), it is good educate 253, middle group No. 3,
It is golden early 47 (early No. 1 of 87-425/ Lu Qing makees parent during the Jinhua institute of agricultural sciences uses, and seed selection is formed, variable rate technology high resistant to rice blast), beautiful
The black paddy of Jiang Xintuan (LTH), former rich morning and C039 are preserved for Rice biology National Key Laboratory of rice in China invention institute.Zhejiang spoke
802nd, good 293, Hunan morning Xian 1, short No. 3 of blue or green paddy and early blue or green No. 1 of the land of educating is rice in China invention institute's Rice Germplasm Resources storehouse Wei Xing
Grey hair bright member provide.The rice blast bacterial strain used in the present invention is collected by this laboratory, separates, identifies and preserved.Lijing is new
The sense susceptible check variety of rice blast high that the black paddy (LTH) of group, former rich morning and C039 are identified as rice blast, early 39 in super early rice
Family tree is as shown in Figure 1.
1.2 Molecular Detections
With reference to Warudeet al. (Warude D, Preeti C, Joshi K, et al.DNA isolation from
fresh and dry plant samples with highly acidic tissue extracts.Plant MolBiol
Rep,2003,21:Method 467a-467f.) extracts paddy rice complete genome DNA, 1% agarose gel electrophoresis and Nano Drop
System, 2000c (Thermo, USA) detect its yield and quality.25 μ L PCR reaction systems and reaction condition are with reference to Liang
et al.(Liang Y,Yan B Y,Peng Y L,et al.Molecular screening for identification
of resistant genes in a germplasm(Oryza sativa L.)collection resistant to
Magnaportheoryzae.Rice Sci, accepted), the annealing temperature of each pair primer is with reference to table 2.By PCR primer loading
In 3% Ago-Gel containing GelRed, electrophoretic separation under BIORAD gel imaging instruments after shooting and preserve picture.PCR is produced
The direct Song Boshang biotechnologys (Shanghai) Co., Ltd. sequencing of thing, each PCR independently carries out expanding simultaneously sample presentation at least 3 times.
Bioinformatic analysis：The DNA sequence dna that sequencing company is sent back to through BioEdit softwares, manually with manual change by check and correction,
And complete sequence assembly.Code area prediction is using online software GeneS can (http://genes.mit.187Edu/
GENSCAN.Html)and Fegenesh(htt p://linux1.softberry.com/berry.phtml188Topic=
Fgenesh＆group=programs＆subgroup=gfind).Sequence alignment using online software MultAli (http:// multalin.toulouse.inra.fr/multalin)。
1.3 inoculated identifications
Rice blast strain number for being inoculated with is 14-754,10-105,12-157,16-754 and 16-161, is 2010-
Abiogenous rice blast disease fringe is gathered from Zhejiang and Jiangxi provinces resend ward field between 2016, conidium separates, training
Supporting and be seeded in rice in China invention institute Experimental Base is carried out.Conidium inoculum density (2-5) × 105Individual/ml, addition
0.2%Tween-20, in rice tillering peak period injection inoculation.Incidence is investigated after one week, according to National agricultural professional standard
(rice blast identification technology specification) records the state of an illness, using the most heavy rice strain of morbidity as the resistance rank of the kind, each weight
As long as have in multiple more than 2 plants it is susceptible be designated as sense (S), be designated as resisting (R) less than 2 plants.
2 results and analysis
2.1 phenotypic evaluations
As shown in figure 1, middle morning 39 is to educate 253 selection cross with good by middle group No. 3.Middle group No. 3 is golden early 47 warp
Somaclonal variation seed selection is formed.Variable rate technology has and the golden early 47 consistent anti-spectrums of rice blast.Rice used by disease-resistant phenotypic evaluation
Seasonal febrile diseases bacterial strain gathers the paddy rice master from Zhejiang Province (14-754,10-105,12-157,16-754) and Jiangxi Province (16-161) respectively
Producing region, in addition to 14-754, is leading Epidemic Races.Material to be tested is inoculated in August in 2016 3, one week later field
Inoculated identification result (table 1) shows：Rice blast biological strain 14-754 infects golden early 47, middle group No. 3 and middle morning 39, and other four
Individual microspecies 10-105,12-157,16-754 and 16-161 show non-affine to golden early 47, middle group No. 3 and middle morning 39, therefore, gold
Early 47,5 biological strains during the anti-spectrum of middle group No. 3 and middle morning 39 is to experiment are completely the same.From for the angle of pedigree, rice blast
Physiological pathology microspecies 14-754 cannot infect the Zhejiang spoke 802 of pedigree upstream, praise and educate 293, Hunan morning Xian 1, the early green grass or young crops of short No. 3 of blue or green paddy and land
No. 1, and infected since golden early 47, fallen ill in middle group No. 3 and middle morning 39.In golden early 47, middle group No. 3 and 39 couples of present invention of middle morning
5 biological strains anti-spectrum it is completely the same.Good as one of the parent of middle morning 39 educates 253 except showing susceptible to 14-754
Outward, the leading Epidemic Races 16-754 in the Zhejiang Province of 2016 also infect it is good educate 253, in explanation early 39 couples of biological strain 14-754 and
The disease-resistant performance of 16-754 rice blast is from middle group No. 3 and golden early 47.
The field phenotypic evaluation of table 1
Field inoculation qualification result in showing early 39 disease-resistant phenotype and ground paddy it is completely the same, because containing in ground paddy material
Pi-d2 and Pi-d3, Pi-d2 the and Pi-d3 allele in explanation in morning 39 cannot assign the resistance to 14-754.
The rice blast resistance gene of table 2 is detected and sequence analysis
2.2 Markers for Detection
As shown in Fig. 2 the specific molecular marker of Pi9 Zhejiang spoke 802, short No. 3 blue or green paddy, the early blue or green No. 1, gold early 47 in land, in
Positive band is detected in group 3 and middle morning 39, and purpose band is not detected in other three materials to be tested.Pid2、
Tetra- specific molecular markers of resistant gene of Pid3, Piz and Pib all detect positive band in 9 materials to be tested.Pi36
It is good educate 293 and it is good educate 253 in be not detected by positive band.Pi64 it is good educate 293 and land it is early it is blue or green No. 1 in be not detected by positive bar
Band.It is worth noting that, gold early 47, middle group No. 3 completely the same with the genotype of middle morning 39, all of resistant gene in the present invention
Molecular labeling can detect positive band.
2.3 resistance gene sequences are analyzed
As shown in figure 3, we first by the DNA sequence dna of Pi-d2 allele in middle morning 39 with its (Pi-d2：
FJ915121) reference sequences (Chen XW, Shang JJ, Chen DX, et al.A B-lectin receptor kinase
gene conferring rice blast resistance.Plant J,2006,46:794-804.) compare, as a result show：
Detect 5 pleomorphism sites altogether compared with reference sequences, in material to be tested, amino acid polymorphisms resistant frequency is 0.00062；Detection
To 3 haplotypes, haplotype polymorphism frequency is 0.556.Using online software " GeneScan (http://
genes.mit.187Edu/GENSCAN.Html)and Fegenesh(http://linux1.softberry.com/
berry.phtml188 Topic=fgenesh＆group=programs＆subgroup=gfind) " to carry out code area pre-
Survey, CDS sequences are converted into amino acid series using BioEdit softwares, with reference amino acid sequence (PID2：ACR15163.1)
Compare, detect two nonsynonymous mutations in material to be tested altogether：D95N and H686R.Wherein D95N is by the aspartic acid in ground paddy B
Sport asparagine, and only detect in golden early 47；And during H686R appears in all of material to be tested, by ground paddy B
Histidine mutagenesis be arginine.
3 discuss
In paddy disease-resistant breeding practice, specifying resistant genotype in disease-resistant parent helps more autotelic to be subject to it
Utilize.The genetic background invention of the blast resisting of centering early 39, contributed to from molecular level understanding resistant gene in middle morning 39
Source and distribution situation, for Rice Resistance To Rice Blast breeding provides theories integration.
In the present invention, Markers for Detection result shows to contain all resistant genes used in the present invention in middle morning 39
Pi9, Pid2, Pid3, Piz, Pi64 and Pib, rice blast resistance of the middle morning 39 with lasting and wide spectrum is explained from one side
The reason for.Golden early 47 and middle group 3 have with the completely the same genotype of middle morning 39, illustrate that the rice blast resistance of these three materials exists
The angle source of molecular labeling is consistent.The result of Field inoculation identification shows that golden early 47, middle group 3 and middle morning 39 are anti-with identical
Spectrum, illustrates that these three materials are identicals in rice blast resistance phenotype.Field inoculation qualification result contains Pi-d2's and Pi-d3
Ground paddy B have with middle group No. 3, gold early 47 and the completely the same anti-spectrum of middle morning 39 are showed rice blast biological strain 14-754 and felt
Disease, it is disease-resistant to other four biological strain performances, illustrate that 14-754 can infect two resistant genes of Pi-d2 and Pi-d3.
Middle morning 39 and spring river, glutinous genetic group positioning result showed that middle morning 39 is contained in the molecular labeling interval where Pi-d2
One main effect blast resistant gene (control by the summertime.Early 39 blast resisting Genetic Mechanisms invention in super early rice.2015, Chinese agriculture
Industry academy of sciences academic dissertation, master).Pi-d2 is positioned on the chromosome of paddy rice the 6th nearly centromere region, connects with RM527 and RM3
Lock, genetic distance is respectively 3.2cM and 3.4cM (Corpet F.Multiple sequence alignment with
hierarchical clustering.Nucl Acids Res,1988,16(22),10881-10890).Pi-d2 is single copy
Gene, one length of coding is 825 transmembrane receptor protein kinases (RLK) of amino acid, and the kinases aminoterminal contains hydrophobicity letter
Number peptide, B-lectin domains, PAN domains and TM domains, c-terminus are typical serine/threonine kinases domain (STK).
Pi-d2 belongs to new disease-resistant gene type, its anti-sense difference caused by a single base mutation, i.e. the of ill-resistant protein
441 amino acid are sported the methionine (M) of susceptible albumen by isoleucine (I).Pi-d2 is constitutive expression, and we use
The method of sequencing has taken Pi-d2 allele in material to be tested.Sequence analysis shows, the Pi-d2 equipotentials in middle morning 39
Gene has a nonsynonymous mutation H686R compared with reference sequences, and this mutation is prevalent in all of material to be tested
In.Three nonsynonymous mutations S321L, I441M and H686R are detected in 35 Asian Cultivated Rices and 6 wild rices, wherein
H686R is detected in all of material to be tested, illustrates that nonsynonymous mutation H686R is generally existing (Li JB, Sun
YD,Liu H,et al.Natural variation of rice blast resistance gene Pi-d2.Genetics
and Molecular Research,2015,14(1):1235-1249).In the present invention, all of material to be tested is at the 441st
Amino acid is isoleucine (I), so being disease-resistant Pid2 allele.During Pyricularia oryzae 14-754 includes material to be tested
Early 39, middle group No. 3, the disease-resistant Pid2 allele of gold early 47 and ground paddy B lose resistance, and Zhejiang spoke 802, good educate 293, Hunan morning Xian
No. 1, blue or green paddy is short No. 3, land it is early it is blue or green No. 1 in there may be other resistant genes, disease-resistant is showed to 14-754.Subsequent experimental can be adopted
The method being combined with molecular marker assisted selection and rice blast biological strain 14-754 inoculated identifications, by this 5 materials
Resistant gene was imported into middle morning 39, to improve its anti-spectrum.Whether disease-resistant Pid2 allele is individually or with other resistances
The early 39 lasting further functional verification experiments of rice blast resistances needs are proved during gene is assigned together.
The present invention is from the sequencing point of field rice blast inoculated identification, rice blast resistance gene Markers for Detection and resistant gene
3 aspects of analysis prove that the disease-resistant performance of rice blast of middle morning 39 couples of biological strains 14-754 and 16-754 organizes No. 3 in passing through, and derives from
Golden early 47.
Described above has shown and described some preferred embodiments of invention, but as previously described, it should be understood that invention is not
Form disclosed herein is confined to, the exclusion to other embodiment is not to be taken as, and can be used for various other combinations, modification
And environment, and can be carried out by the technology or knowledge of above-mentioned teaching or association area in invention contemplated scope described herein
Change.And the change and change that those skilled in the art are carried out do not depart from the spirit and scope of invention, then all should be in the appended power of invention
In the protection domain that profit is required.
SEQUENCE LISTING
<110>China Paddy Rice Inst
<120>A kind of method of early 39 rice blast resistance genes in identification super early rice
<130> 2016
<160> 14
<170> PatentIn version 3.5
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Claims (2)
1. a kind of method for identifying early 39 rice blast resistance genes in super early rice, it is characterised in that comprise the steps：
1) with the rice varieties in the rice blast inoculation ground paddy B, middle morning 39 and its pedigree that infect middle morning 39；
2) DNA extracted using pid2 primer pairs enters performing PCR amplification；
3) pcr amplification product is sequenced, and through BioEdit softwares, manually check and correction is repaiied with manual by the DNA sequence dna after sequencing
Change, and complete sequence assembly, prepare the paddy rice sample DNA sequence containing pid2 allele；By pid2 allele pieces
Section is converted to amino acid sequence；
4) result interpretation：
By the amino acid sequence of pid2 allele fragments and reference amino acid sequence (PID2：ACR15163.1) it is compared；
If the amino acid series of pid2 allele have a nonsynonymous mutation I441M, i.e., its 441st amino acid is by different bright
Histidine mutations are the methionine of susceptible albumen, then the paddy rice sample to be detected is the super morning with susceptible Pid2 allele
Early 39 kinds in rice.
2. in identification super early rice according to claim 1 early 39 rice blast resistance genes method, it is characterised in that
Pid2 primers include pid2F primer and nucleotide sequence of the nucleotide sequence as shown in SEQ ID No.1 such as SEQ ID No.2 institutes
The pid2R primers for showing.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107858446A (en) * | 2017-12-21 | 2018-03-30 | 辽宁省盐碱地利用研究所 | A kind of molecular labeling, authentication method and application for identifying Rice Resistance To Rice Blast |
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| CN107988337A (en) * | 2017-11-06 | 2018-05-04 | 云南省农业科学院生物技术与种质资源研究所 | A kind of labeling method and its application of rice blast resistant rice identification method and gene |
| CN107988337B (en) * | 2017-11-06 | 2021-04-13 | 云南省农业科学院生物技术与种质资源研究所 | Identification method of rice blast resistance rice, gene marking method and application thereof |
| CN107858446A (en) * | 2017-12-21 | 2018-03-30 | 辽宁省盐碱地利用研究所 | A kind of molecular labeling, authentication method and application for identifying Rice Resistance To Rice Blast |
| CN109468400A (en) * | 2018-12-07 | 2019-03-15 | 袁隆平农业高科技股份有限公司 | Rice blast resistant gene Pi36 codominant marker and application |
| CN109468400B (en) * | 2018-12-07 | 2022-03-22 | 袁隆平农业高科技股份有限公司 | Rice blast resistance gene Pi36 codominant molecular marker and application thereof |
| CN111304218A (en) * | 2020-03-20 | 2020-06-19 | 西南大学 | Rice resistance gene OsRLR1 and application thereof in rice blast resistance |
| CN111304218B (en) * | 2020-03-20 | 2022-08-16 | 西南大学 | Rice resistance gene OsRLR1 and application thereof in rice blast resistance |
| CN113903397A (en) * | 2021-08-23 | 2022-01-07 | 华南农业大学 | Technical system with inclusion and accurate identification and excavation of rice blast Pib disease-resistant gene family functional genes |
| CN113903397B (en) * | 2021-08-23 | 2022-09-02 | 华南农业大学 | Method for identifying and mining rice blast Pib disease-resistant gene family functional genes with inclusion and precision and application thereof |
| CN113789403A (en) * | 2021-09-02 | 2021-12-14 | 华南农业大学 | Three sets of compatible and accurate rice blast Pid disease-resistant gene family allele identifying and mining technical systems |
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