CN106834485A - A kind of molecular labeling of Rice Resistance seasonal febrile diseases gene Pi9 and its application - Google Patents
A kind of molecular labeling of Rice Resistance seasonal febrile diseases gene Pi9 and its application Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 47
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 45
- 235000009566 rice Nutrition 0.000 title claims abstract description 42
- 238000002372 labelling Methods 0.000 title claims abstract description 18
- 208000015220 Febrile disease Diseases 0.000 title claims abstract description 17
- 230000001932 seasonal effect Effects 0.000 title claims abstract description 14
- 240000007594 Oryza sativa Species 0.000 title abstract description 4
- 241000209094 Oryza Species 0.000 claims abstract description 41
- 238000004519 manufacturing process Methods 0.000 claims abstract description 7
- 238000012408 PCR amplification Methods 0.000 claims abstract description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 claims abstract description 6
- 230000002068 genetic effect Effects 0.000 claims abstract description 4
- 238000002474 experimental method Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 10
- 238000009395 breeding Methods 0.000 abstract description 9
- 238000000034 method Methods 0.000 abstract description 9
- 230000001488 breeding effect Effects 0.000 abstract description 8
- 239000003550 marker Substances 0.000 abstract description 5
- 201000010099 disease Diseases 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 241000196324 Embryophyta Species 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 208000035240 Disease Resistance Diseases 0.000 description 2
- 241001330975 Magnaporthe oryzae Species 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 241001344131 Magnaporthe grisea Species 0.000 description 1
- 240000000125 Oryza minuta Species 0.000 description 1
- 101150050192 PIGM gene Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 238000003976 plant breeding Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
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- C12Q2600/13—Plant traits
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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Abstract
Molecular labeling and its application the present invention relates to a kind of Rice Resistance seasonal febrile diseases gene Pi9, it is with oryza sativa genomic dna as template using the method for molecular labeling I9 assist-breeding anti-rice blast rices, I9 enters performing PCR amplification for primer, amplified production is detected by 2% agarose gel electrophoresis, if the single slice of 565bp, then it represents that the detection sample is Pi9 gene pures；If the single slice of 688bp, then it represents that the detection sample is free of Pi9 genes；If 565bp and the bands of 688bp two, then it represents that the detection sample is Pi9 genetic heterozygosis.The mark is designed by sequence difference and exchanged in the absence of heredity, and accuracy is high；Agarose gel electrophoresis detection is directly used in, it is more easy；It is codominant marker, homozygosis and heterozygous individual can be detected, shorten breeding cycle, improves efficiency.
Description
Technical field
The present invention relates to molecular genetics field, and in particular to a kind of molecular labeling of Rice Resistance seasonal febrile diseases gene Pi9 and its
Using.
Background technology
Used as most important cereal crops, the whole world exceedes the population of more than half with paddy rice as staple food to paddy rice, by ascus
The rice blast that bacterium [Magnaporthe grisea (Hebert) Barr] causes is most serious in Rice Production in world wide
One of disease, disease can occur each growth period in paddy rice, general underproduction 10%-20%, when serious even particle without
Receive, be one of restriction factor of world food safety.Chemical prevention and control can mitigate disease, reduce production loss to a certain extent,
But a large amount of due to chemical pesticide pollute using meeting to environment.Therefore, seed selection and plantation anti-rice blast rice kind are most
Measure economic, most effective, most friendly to environment.Due to rice blast biological strain pathogenicity variation frequently, single resistant variety
Often resistance against diseases will be lost gradually after several years of popularization and application, so excavating, being to obtain lasting using broad-spectrum resistance gene
The important channel of disease-resistant variety.
Rice blast resistance is typical quantitative character, by controlled by multiple genes and easily affected by environment.Conventional breeding for disease resistance
In, Disease Resistance Identification result is inaccurate to cause breeding efficiency relatively low.Molecular marker assisted selection is a kind of utilization and resistant gene
Functional label in close linkage mark or gene, combines genotype and phenotypic evaluation is selected objective trait in offspring
GENERALIZATION OF MODERN BREEDING TECHNIQUE.The method is not only substantially shorter the breeding time limit, improves breeding efficiency, and also a saving substantial amounts of people
Power, material resources cost.Pi9 is a broad spectrum antidisease gene from Oryza minuta, and silk is close to positioned at Short arm of chromosome 6
The Pi2/9 sites of grain position, with multiple disease-resistant gene equipotentials such as Pi2, Piz-t, Pigm, Pi40, Pi50, Pi2-2, research shows
Pi9 genes are at least resistant to 21 physiological races of rice blast fungus, there is very big application value in terms of blast resisting breeding,
The functional indicia of Pi9 genes is developed to making full use of the gene, further improvement Rice Resistance To Rice Blast important in inhibiting.
The content of the invention
The technical problem to be solved in the present invention is：Propose a kind of molecular labeling of rice blast resistant gene Pi9, its just
Anti-primer and its application.
The present invention is that the technical scheme for solving above-mentioned technical problem proposition is：A kind of molecule of Rice Resistance seasonal febrile diseases gene Pi9
Mark, the molecular labeling numbering is I9, and the positive and negative primer sequence of the molecular labeling I9 is：
I9 F：5’- ATTGTAATTCTAGCCTTCCGTCC -3’
I9 R：5’- TCCATTTCAGAAACAGAGCACA -3’.
The invention provides the purposes of molecular labeling I9：The molecular labeling I9 is used for the mirror of Rice Resistance seasonal febrile diseases gene Pi9
The assisted selection of fixed and/or anti-seasonal febrile diseases paddy rice.
Further, the identification for Rice Resistance seasonal febrile diseases gene Pi9 includes following experimental procedure：
I, extraction are for examination paddy rice sample gene group；
Ii, using the molecular labeling I9 positive and negative primer combination to is extracted in step i genome enter performing PCR amplification, acquisition
Amplified production；
Iii, the amplified production is detected by 2% agarose gel electrophoresis, if the single slice of 565bp, then it represents that described
Sample is Pi9 gene pures；If the single slice of 688bp, then it represents that the sample is free of Pi9 genes；If 565bp and
The bands of 688bp two, then it represents that the sample is Pi9 genetic heterozygosis.
The beneficial effects of the invention are as follows：
The present invention designs the Functional marker of Pi9 genes by using nucleotide sequence difference, is exchanged in the absence of heredity, accurately
Property is high；Agarose gel electrophoresis detection is can be directly used for because amplified fragments differ greatly, it is more easy；It is codominant marker,
Homozygosis and heterozygous individual can be detected, shortens breeding cycle, improve efficiency.
Brief description of the drawings
Molecular labeling and its application below in conjunction with the accompanying drawings to a kind of Rice Resistance seasonal febrile diseases gene Pi9 of the invention makees further
Explanation.
Fig. 1 be in the present invention molecule I9 marks to 8 electrophoresis detection figures of rice varieties.
Note：Wherein 1-8 represent respectively rice varieties Shanghai drought 1B, elegant water 123, Shanghai drought 7B, Huang Huazhan, middle group 14, Shanghai it is non-irrigated
11B, Nipponbare and 75-1-127；M represents D2000 Marker.
Specific embodiment
Embodiment 1：The exploitation of Pi9 gene molecule markers
（1）, material to be tested：Disease resisting rice parent：75-1-127；Susceptible rice varieties：Shanghai drought 1B, elegant water 123, Shanghai drought 7B is yellow
China accounts for, middle group 14, Shanghai drought 11B, Nipponbare.
（2）, oryza sativa genomic dna extracting method refer to Lou Qiaojun（2006）Described in method.（Lou Qiaojun, Chen Liang, sieve
The tri- kinds of comparing of oryza sativa genomic dna rapid extracting method [J] Molecular Plant Breedings of sharp army, 2006,3 (5):
749-752）
（3）, gene molecule marker I9 exploitation：Pi9 is a wide spectrum blast resistant gene cloned, positioned at the 6th chromosome
The Pi2/9 sites of galianconism, the whole genome sequence of Pi9 comes from GenBank databases, and indexed number is DQ285630, in Nipponbare
The susceptible gene order indexed number of equipotential in reference gene group is DQ454158, between the 13239 to 13476th base, 75-
1-127 is compared with Nipponbare and has been lacked 124 bases, is shown in Table 1.
Table 1：The Base comparison of Nipponbare and 75-1-127
| Nipponbare | CTCAATCTCCTTGGCGATGGAAGTTTTCTTAATATTGTAGGCACCACATTTCTAGCTGAT |
| 75-1-127 | CTCAATCTC-------------------------TTGCAGGC--TGAGTTGGTAGC---- |
| ********* *** **** ** **** | |
| Nipponbare | AATGCAACTATCGTGGAGATCATAAAGAGGAGGAATTTTGTGGAAGAAGGACCTGGAGGT |
| 75-1-127 | AATGCAATT-----GGCGAT----------GGGAGTTTT-------------CTTAATAT |
| ******* * ** *** *** **** ** * * | |
| Nipponbare | TGGGAGTCTTCGGCCTCTACTCAGTCAAATACAAGAAAATATTCCTCAAAACTTAATGCA |
| 75-1-127 | TG-------TAGGCATC---------------------ACATTTCT-AACACACAATGCA |
| ** * *** ** * *** ** ** ** ****** | |
| Nipponbare | TGTGATGTAGATTCAAAAAGAGGTGAATCAACTAGCTGATAAGTTAGCAAAGGATGTGAG |
| 75-1-127 | -------------------ACGGTGTATCGACTAGGTGATAAGGTAGCAAAGGATGTGAG |
| **** *** ***** ******* **************** | |
| Nipponbare | ATCGCGACAGCTAAATCCTTCAGGTTTATGATTGTCAAAATATCTTGAGTCTTGCTCATC |
| 75-1-127 | ATCGCAACAGCTCAATCCTTCAG-TTTATGATTGTCAAAATATCTTGAGTCTTGCTCACC |
| ***** ****** ********** ********************************** * | |
| Nipponbare | CTAGAAACCTTTGTTATGTTAGAGATTTGAAGAACCATTTCAGAGCAACGAATTGTATTA |
| 75-1-127 | CTAGAAACCTTCGTTATGCTTGAGATTTGAAAAA--------GAGCAACGAATTGTATTA |
| *********** ****** * ********** ** ****************** | |
| Nipponbare | CAACTTTACAAGTCATGTGCTCTGTTTCTGAAATGGAAATAAAACTGATGAGTTCTAAAA |
| 75-1-127 | C--------AAGTCATGTGCTCTGTTTCTGAAATGGAAATAATACTGACGAGTTCTAAAG |
| * ********************************* ***** ********** |
（4）, according to nucleotide sequence difference, using the software Design primers I9 of Primer Premier 5.0, its positive anti-primer sequence
It is classified as：
I9 F：5’- ATTGTAATTCTAGCCTTCCGTCC -3’
I9 R：5’- TCCATTTCAGAAACAGAGCACA -3’.
Embodiment 2：Parent's polymorphic detection of Pi9 gene molecule markers
（1）, extract for examination rice varieties genome, extracting method be routine CTAB methods.It is respectively for examination rice varieties：Shanghai drought
1B, elegant water 123, Shanghai drought 7B, Huang Huazhan, middle group 14, Shanghai drought 11B, Nipponbare and 75-1-127.
（2）, expanded to entering performing PCR for examination rice varieties using functional label I9, PCR amplification system is：20ng paddy rice bases
Because of a group DNA profiling, 10 uL Taq PCR Mastermix（TIANGEN Biotech (Beijing) Co., Ltd.）, draw before and after 10 uM
Each 1 uL of thing, supplement dd H2O to 20 uL.PCR response procedures are：95 DEG C of min of predegeneration 5, then by 95 DEG C of 30 s, 55
30 s at DEG C, 1 min carries out 30 circulations at 72 DEG C, and 5 min are extended at last 72 DEG C.
（3）, take the uL of PCR primer 8 be splined on 2% agarose gel electrophoresis detection.Result is as shown in figure 1, disease resisting rice
Parent 75-1-127 amplifies the single slice of 565bp, and the single slice of 688bp is amplified in other susceptible rice varieties, electricity
Swimming band is clear, and difference is obvious.
Embodiment 3：The checking of Pi9 gene molecule markers and application
Using Pi9 gene molecule markers I9 to the BC3F1 of paddy rice maintainer Shanghai drought 1B and disease-resistant parent 75-1-127 for colony
Genomic DNA enters performing PCR amplification, and product carries out electropherotyping, detects 33 samples and contain 565bp in 2% Ago-Gel
With the bands of 688bp two, represent that these detection samples are Pi9 genetic heterozygosis.Artificial infection Pyricularia oryzae qualification result shows that this 33
Individual individual plant resists to disease-resistant level in showing as.
350 yellow China are accounted for and 75-1-127 F using Pi9 gene molecule markers I92The genomic DNA of individual plant enters performing PCR
Amplification, product carries out electrophoresis detection, there is 90 Pi9 gene pures individual plants, 169 Pi9 genes respectively in 2% Ago-Gel
Heterozygosis individual plant and 91 are free of Pi9 gene individual plants, and in Jinggang Mountain, natural rice blast qualification result shows, the individual plant containing Pi9 genes
Disease-resistant level is shown, the individual plant without Pi9 genes shows susceptible phenotype.
Therefore show can using the mark carry out Rice Resistance seasonal febrile diseases gene Pi9 identification and/or anti-seasonal febrile diseases paddy rice it is auxiliary
Help selection and use.
Of the invention to be not limited to above-described embodiment, the technical scheme of above-mentioned each embodiment of the invention can be handed over each other
Fork combination forms new technical scheme, and the technical scheme that all use equivalents are formed in addition all falls within the guarantor of application claims
In the range of shield.
Claims (3)
1. a kind of molecular labeling of Rice Resistance seasonal febrile diseases gene Pi9, it is characterised in that the molecular labeling numbering is I9, described point
Son mark I9 positive and negative primer sequence be：
I9 F：5’- ATTGTAATTCTAGCCTTCCGTCC -3’
I9 R：5’- TCCATTTCAGAAACAGAGCACA -3’.
2. the molecular labeling of Rice Resistance seasonal febrile diseases gene Pi9 according to claim 1, it is characterised in that：The molecular labeling I9
The assisted selection of identification and/or anti-seasonal febrile diseases paddy rice for Rice Resistance seasonal febrile diseases gene Pi9.
3. the molecular labeling of Rice Resistance seasonal febrile diseases gene Pi9 according to claim 2, it is characterised in that described for Rice Resistance
The identification of seasonal febrile diseases gene Pi9 includes following experimental procedure：
I, extraction are for examination paddy rice sample gene group；
Ii, using the molecular labeling I9 positive and negative primer combination to is extracted in step i genome enter performing PCR amplification, acquisition
Amplified production；
Iii, the amplified production is detected by 2% agarose gel electrophoresis, if the single slice of 565bp, then it represents that described
Sample is Pi9 gene pures；If the single slice of 688bp, then it represents that the sample is free of Pi9 genes；If 565bp and
The bands of 688bp two, then it represents that the sample is Pi9 genetic heterozygosis.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146695A (en) * | 2013-03-01 | 2013-06-12 | 福建省农业科学院生物技术研究所 | Functional molecular marker for rice anti-blast gene Pi9 and application thereof |
| CN103305510A (en) * | 2013-07-10 | 2013-09-18 | 广东省农业科学院植物保护研究所 | Rice blast resistance gene Pi9 gene specificity molecular marker Pi9SNP as well as preparation and application thereof |
| CN105950743A (en) * | 2016-05-31 | 2016-09-21 | 淮阴师范学院 | Rice blast resistance gene Pi9 functional specificity molecular marker and application thereof |
| CN106434869A (en) * | 2016-08-04 | 2017-02-22 | 上海市农业生物基因中心 | Brown-planthopper-resistant gene Bph14 molecular marker for rice and application |
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2017
- 2017-02-28 CN CN201710110776.2A patent/CN106834485B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146695A (en) * | 2013-03-01 | 2013-06-12 | 福建省农业科学院生物技术研究所 | Functional molecular marker for rice anti-blast gene Pi9 and application thereof |
| CN103305510A (en) * | 2013-07-10 | 2013-09-18 | 广东省农业科学院植物保护研究所 | Rice blast resistance gene Pi9 gene specificity molecular marker Pi9SNP as well as preparation and application thereof |
| CN105950743A (en) * | 2016-05-31 | 2016-09-21 | 淮阴师范学院 | Rice blast resistance gene Pi9 functional specificity molecular marker and application thereof |
| CN106434869A (en) * | 2016-08-04 | 2017-02-22 | 上海市农业生物基因中心 | Brown-planthopper-resistant gene Bph14 molecular marker for rice and application |
Non-Patent Citations (10)
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