CN107034308A - The molecular labeling of Rice Resistance seasonal febrile diseases gene Pigm a kind of and its application - Google Patents
The molecular labeling of Rice Resistance seasonal febrile diseases gene Pigm a kind of and its application Download PDFInfo
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- CN107034308A CN107034308A CN201710502112.0A CN201710502112A CN107034308A CN 107034308 A CN107034308 A CN 107034308A CN 201710502112 A CN201710502112 A CN 201710502112A CN 107034308 A CN107034308 A CN 107034308A
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Abstract
The present invention relates to the molecular labeling of Rice Resistance seasonal febrile diseases gene Pigm a kind of and its application, it is using oryza sativa genomic dna as template using the method for the assist-breeding anti-rice blast rices of molecular labeling gm 4, gm 4 is that primer enters performing PCR amplification, amplified production is detected by 2% agarose gel electrophoresis, if 516bp band, then it represents that detection sample Pigm containing blast resistant gene.Heredity exchange is not present by sequence difference design in the mark, and accuracy is high；Agarose gel electrophoresis detection is directly used in, it is more easy；For codominant marker, homozygosis and heterozygous individual can be detected, shortens breeding cycle, efficiency is improved.
Description
Technical field
The present invention relates to molecular genetics field, and in particular to the molecular labeling of Rice Resistance seasonal febrile diseases gene Pigm a kind of and its
Using.
Background technology
The rice blast as caused by sac fungus [Magnaporthe grisea (Hebert) Barr] is paddy rice in world wide
Each growth period in paddy rice can occur for one of disease of most serious in production, disease, underproduction 40%-50% when serious,
Even No kernels or seeds are gathered, as in a year of scarcity, is one of the restriction factor of world food safety.It is a large amount of that environment is polluted using chemical pesticide, control
The most economical effective method of this disease is excavation and carries out seed selection broad-spectrum disease resistance new product using new broad spectrum durable Resistant germplasm
Kind.
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 utilizes and resistant gene
Functional label in close linkage mark or gene, combines genotype in offspring and phenotypic evaluation is selected objective trait
GENERALIZATION OF MODERN BREEDING TECHNIQUE.This 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.Pigm is a wide spectrum blast resistant gene, than generally acknowledged broad-spectrum resistance gene Pi1, Pi2, Pi3 anti-spectrum
It is wider；Pigm derives from Sichuan landraces paddy plum No. 4, in the 30 strong pathogenic strain of various regions and nations
29 performances are high anti-or immune, and in 2017 by successful clone (Deng et al., 2017), Pigm Jie is explained from mechanism
That leads is disease-resistant with persistence, and final production is not influenceed again using the Pigm existing broad spectrum durable disease resistances of kind for improveing seed selection
Amount, there is very big application value in terms of blast resisting breeding, develops the functional indicia of Pigm genes to making full use of the base
Cause, further improves 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 rice blast resistant gene Pigm molecular labeling, its
Positive anti-primer and its application.
The present invention is that the technical scheme for solving above-mentioned technical problem proposition is：A kind of point of Rice Resistance seasonal febrile diseases gene Pigm
Son mark, the molecular labeling numbering is gm-4, and the positive and negative primer sequence of the molecular labeling gm-4 is：
gm-4F：5’-CTTGTCTCTCTCGCCCTCTCA-3’
gm-4R：5’-AGGAGTCAGGACTTAGGGTTTCT-3’.
The invention provides molecular labeling gm-4 purposes：The molecular labeling gm-4 is used for Rice Resistance seasonal febrile diseases gene Pigm
Identification and/or anti-seasonal febrile diseases paddy rice assisted selection.
Further, the identification for Rice Resistance seasonal febrile diseases gene Pigm includes following experimental procedure：
I, extraction are for examination paddy rice sample gene group；
Ii, using the molecular labeling gm-4 positive and negative primer combination to extracted in step i genome enter performing PCR expansion
Increase, obtain amplified production；
Iii, the amplified production detected by 2% agarose gel electrophoresis, if 515bp single slice, then table
It is Pigm gene pures to show the sample.
The beneficial effects of the invention are as follows：
The present invention designs the Functional marker of Pigm genes by using nucleotide sequence difference, in the absence of heredity friendship
Change, accuracy is high；Agarose gel electrophoresis detection is directly used in, it is more easy, carry out anti-rice blast rice using the mark auxiliary
Help selection to shorten 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 Pigm of the invention is made into one
Walk explanation.
Fig. 1 is electrophoresis detection figure of the molecule gm-4 marks to 23 rice varieties in the present invention.
Note：1-23 represents rice varieties 1 respectively：Extensive No. 3 of drought, 2：BL6,3：Shanghai drought 1B, 4：B5,5：SAGC4,6：Japan
It is fine, 7：Huang Huazhan, 8：603B, 9：0412,10：389B, 11：R9,12：Magnificent 15B, 13：R15,14：Shanghai drought 9B, 15：R37,16：
Shanghai drought 11B, 17：N632S, 18：Elegant water 123,19：R49,20：Hunan is fine, and 21：Shanghai drought 7B, 22：75-1-127,23：Paddy plum No. 4.M
Represent D2000Marker.
Embodiment
Embodiment 1：The exploitation of Pigm gene molecule markers
(1), material to be tested.
Disease resisting rice kind：Paddy plum No. 4.
Susceptible rice varieties：1：Extensive No. 3 of drought, 2：BL6,3：Shanghai drought 1B, 4：B5,5：SAGC4,6：Nipponbare, 7：Huang Huazhan,
8：603B, 9：0412,10：389B, 11：R9,12：Magnificent 15B, 13：R15,14：Shanghai drought 9B, 15：R37,16：Shanghai drought 11B, 17：
N632S, 18：Elegant water 123,19：R49,20：Hunan is fine, and 21：Shanghai drought 7B, 22：75-1-127.
(2), oryza sativa genomic dna extracting method method with reference to described in Lou Qiaojun (2006).(Lou Qiaojun, Chen Liang, sieve
Comparison [J] Molecular Plant Breedings of sharp tri- kinds of Rapid Methods of DNA Extraction from Rice of army, 2006,3 (5):749-752)
(3), gene molecule marker gm-4 exploitation：Pigm is the wide spectrum a cloned blast resistant gene, positioned at the 6th
Chromosome, and Pi-9, Pi-z, the relation of the equipotential each other such as Pi2, Pigm whole genome sequence comes from GenBank databases, received
Record number is KU904633.2, compares and finds, 876bp fragment of the Pigm genes from the 92245 to 93120th base in Gu Mei 4
(being shown in Table 1) and this fragment being missing from the corresponding susceptible allele of reference gene group Nipponbare (DQ454158.1) may be used also
To be matched with the 12nd chromosome (21574111bp-21573243bp), therefore individually expand this fragment and can not accurately judge whether
Containing Pigm genes, thus before this fragment design a forward primer, one reverse primer of fragment medium design, containing
Have in the disease-resistant variety of Pigm genes can positive anti-primer can match amplification, and in the susceptible variety without Pigm genes can not
With can not expand.
Table 1：The number of base of Nipponbare and paddy plum No. 4 is compared
Explanation：* number identical base is marked out ,-number represent missing base.
(4), according to nucleotide sequence difference, using the software Design primers gm-4 of Primer Premier 5.0, its is positive and negative
Primer sequence is：
gm-4F：5’-CTTGTCTCTCTCGCCCTCTCA-3’
gm-4R：5’-AGGAGTCAGGACTTAGGGTTTCT-3’.
Embodiment 2：Parent's polymorphic detection of Pigm gene molecule markers
(1), extract for examination rice varieties genome, extracting method is routine CTAB methods.It is respectively for examination rice varieties：1：
Extensive No. 3 of drought, 2：BL6,3：Shanghai drought 1B, 4：B5,5：SAGC4,6：Nipponbare, 7：Huang Huazhan, 8：603B, 9：0412,10：389B,
11：R9,12：Magnificent 15B, 13：R15,14：Shanghai drought 9B, 15：R37,16：Shanghai drought 11B, 17：N632S, 18：Elegant water 123,19：R49,
20：Hunan is fine, and 21：Shanghai drought 7B, 22：75-1-127、23：No. 4 Shanghai drought 1B of paddy plum, elegant water 123, Shanghai drought 7B, Huang Huazhan, middle group 14,
Shanghai drought 11B, Nipponbare and 75-1-127.
(2), using functional label gm-4 to entering performing PCR amplification for examination rice varieties, PCR amplification system is：20ng paddy rice
Genomic DNA template, 10uL Taq PCR Mastermix (TIANGEN Biotech (Beijing) Co., Ltd.), 10uM's is front and rear
Each 1uL of primer, supplements dd H2O to 20uL.PCR response procedures are：95 DEG C of pre-degeneration 5min, then by 95 DEG C of 30s, at 58 DEG C
30s, 1min carries out extending 5min at 30 circulations, last 72 DEG C at 72 DEG C.
(3) PCR primer 8uL, is taken to be splined on 2% agarose gel electrophoresis detection.As a result as shown in figure 1, disease resisting rice
The single slice that parent Gu Mei 4 amplifies 516bp, can not expand the band for obtaining 516bp in other susceptible rice varieties,
Electrophoretic band is clear, and difference is obvious.
Embodiment 3：The checking and application of Pigm gene molecule markers
To carry the disease-resistant parent Gu Mei 4 of Pigm genes for donor, extensive No. 3 of saving water, resisting drought rice restorer drought is acceptor,
The rice blast resistance of extensive No. 3 of improvement drought.The BC1F2 of extensive No. 3/Gu Mei 4 // extensive No. 3 of the drought of drought is for colony's totally 384 single-strain plantings
Base is identified in Jinggang Mountain natural rice blast, first with genomic DNAs of the Pigm gene functions mark gm-4 to 384 individual plants
Enter performing PCR amplification, product carries out electropherotyping in 2% Ago-Gel, and detecting 280 individual plants has 516bp band, table
Show that these individual plants contain Pigm genes, remaining 104 individual plants do not amplify 3 on 516bp bands, coincidence statistics：1
Ratio.Blast resistance identification result shows that 280 individual plants of the gene containing Pigm show disease-resistant level, without Pigm
The individual plant of gene shows susceptible phenotype.
Therefore show can using mark progress Rice Resistance seasonal febrile diseases gene Pigm identification and/or anti-seasonal febrile diseases paddy rice it is auxiliary
Help selection and use.
The present invention's is not limited to above-described embodiment, and the technical scheme of each above-mentioned embodiment of the invention can be handed over each other
Fork combination forms new technical scheme, and the technical scheme of all use equivalent substitution formation, all falls within the guarantor of application claims in addition
In the range of shield.
Claims (3)
1. a kind of Rice Resistance seasonal febrile diseases gene Pigm molecular labeling, it is characterised in that the molecular labeling numbering is gm-4, described
Molecular labeling gm-4 positive and negative primer sequence is：
gm-4 F：5’- CTTGTCTCTCTCGCCCTCTCA -3’
gm-4 R：5’- AGGAGTCAGGACTTAGGGTTTCT -3’.
2. Rice Resistance seasonal febrile diseases gene Pigm molecular labeling according to claim 1, it is characterised in that：The molecular labeling
Gm-4 is used for Rice Resistance seasonal febrile diseases gene Pigm identification and/or the assisted selection of anti-seasonal febrile diseases paddy rice.
3. Rice Resistance seasonal febrile diseases gene Pigm molecular labeling according to claim 2, it is characterised in that described to be used for Rice Resistance
Seasonal febrile diseases gene Pigm identification includes following experimental procedure：
I, extraction are for examination paddy rice sample gene group；
Ii, using the molecular labeling gm-4 positive and negative primer combination to extracted in step i genome enter performing PCR expand, obtain
Obtain amplified production；
Iii, the amplified production detected by 2% agarose gel electrophoresis, if 515bp single slice, then it represents that described
Sample is Pigm gene pures.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628627A (en) * | 2018-12-11 | 2019-04-16 | 华智水稻生物技术有限公司 | The SNP marker development and application of broad-spectrum rice-blast resistant gene of paddy rice Pigm |
| CN110408719A (en) * | 2019-08-05 | 2019-11-05 | 江苏省农业科学院 | A kind of four primer molecule labeling methods for identifying rice blast resistant gene Pigm |
| CN112410459A (en) * | 2020-12-11 | 2021-02-26 | 上海市农业生物基因中心 | KASP molecular marker for detecting rice blast resistance gene Pi25 and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628627A (en) * | 2018-12-11 | 2019-04-16 | 华智水稻生物技术有限公司 | The SNP marker development and application of broad-spectrum rice-blast resistant gene of paddy rice Pigm |
| CN109628627B (en) * | 2018-12-11 | 2022-03-18 | 华智生物技术有限公司 | Development and application of SNP (single nucleotide polymorphism) marker of broad-spectrum rice blast resistance gene Pigm of rice |
| CN110408719A (en) * | 2019-08-05 | 2019-11-05 | 江苏省农业科学院 | A kind of four primer molecule labeling methods for identifying rice blast resistant gene Pigm |
| CN112410459A (en) * | 2020-12-11 | 2021-02-26 | 上海市农业生物基因中心 | KASP molecular marker for detecting rice blast resistance gene Pi25 and application thereof |
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