CN102162011A - Molecule marking method of rice blast-resisting gene - Google Patents
Molecule marking method of rice blast-resisting gene Download PDFInfo
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- CN102162011A CN102162011A CN2011101188194A CN201110118819A CN102162011A CN 102162011 A CN102162011 A CN 102162011A CN 2011101188194 A CN2011101188194 A CN 2011101188194A CN 201110118819 A CN201110118819 A CN 201110118819A CN 102162011 A CN102162011 A CN 102162011A
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Abstract
The invention discloses a molecule marking method of a rice blast-resisting gene, belonging to the field of crop molecule heredity and breeding. The method comprises the following steps: (1) taking a rice sample and extracting a genome DNA (Deoxyribose Nucleic Acid) of the rice sample; and (2) carrying out PCR (Polymerase Chain Reaction) amplification on the genome DNA of the rice sample by utilizing any one pair of molecule-marked primers in RM6091 and RM26632, carrying out electrophoresis detection on a PCR amplification product, and if a molecule-marked DNA segment with corresponding size is amplified, showing that a Pi-bdl(t) gene exists. Through rice blast-resisting gene Pi-bdl(t) molecule marking in the invention, whether thin rice as well as crossbred descendants, backcross descendants and multiple cross descendants thereof contain the gene can be detected, the resistance level of the gene on rice blast can be forecasted, the selecting efficiency of rice blast resistant materials can be greatly increased, and the breeding process for disease resistance can be accelerated.
Description
Technical field
The invention belongs to farm crop molecular genetic thremmatology field, relate to the molecule marking method of rice blast resistant gene.
Background technology
Paddy rice is one of most important food crop of China, and is significant to ensureing China's grain security and growth of the national economic.Rice blast is severe diseases on China's Rice Production, have propagate fast, take place wide, harm heavily wait characteristics (Ling Zhongzhuan etc., 1989, northern China rice district physiological races of rice blast fungus is studied towards state's agricultural sciences, 22 (3): 7-13).Further excavate and utilize China's blast resistant gene resource, cultivating and plant disease-resistant variety is that control rice blast takes place and the minimizing rice yield loses most economical valid approach.
So far, the various countries scientist has identified more than 70 blast resistant gene from paddy rice, wherein has 17 disease-resistant genes successfully to be cloned.Modern varieties be introduced or be aggregated to these disease-resistant genes can, selects kind high anti-, wide spectrum.But traditional breeding way is time-consuming, effort, phenotypic evaluation difficulty, breeding efficiency are low, because disease-resistant gene mostly is and often exists epistasis to do mutually between dominance, gene, the disease-resistant gene polymerization is more difficult.Can effectively address this problem by molecular mark.
China Taihu Lake basin rice does with a long history, be considered to one of japonica rice area of origin, contain abundant seed rice resource is arranged, Li Peifu etc. (1999, the genetic research of two Taihu Lake basin japonica rice local variety blast resistings. the rice in China science, 13 (1): 11-14) the thin rice of report China Taihu Lake basin japonica rice local variety is to the resistance performance wide spectrum and the high anti-characteristics of Pyricularia oryzae.Identify from the distinctive germ plasm resource of China and clone's blast resistant gene that the gene that can obtain to have independent intellectual property right is of great importance to the blast resisting breeding that improves China rice anti-rice blast breeding level, especially japonica rice.
Summary of the invention
The objective of the invention is: the molecule marking method that the thin rice blast resistant gene Pi-bd1 (t) of rice varieties is provided.By detecting and the closely linked molecule marker of disease-resistant gene Pi-bd1 (t), can define no disease-resistant gene Pi-bd1 (t), and the prediction rice plant rice blast resistance, the seed selection progress of quickening anti-rice blast rice new variety.
Purpose of the present invention can be achieved through the following technical solutions:
The molecule marking method of the thin rice blast resistant gene Pi-bd1 (t) of paddy rice comprises following operation steps:
(1) water intaking rice sample extracts paddy rice sample gene group DNA;
(2) utilize any a pair of in the table 1 or two pairs and the thin closely linked molecule marker primer of rice blast resistant gene Pi-bd1 (t) of paddy rice, described paddy rice sample gene group DNA is carried out pcr amplification, pcr amplification product carries out electrophoresis detection on 8% non-denaturing polyacrylamide gel, if amplify the molecular marker DNA fragment of corresponding size, indicate the existence of Pi-bd1 (t) gene:
Table 1
Wherein, the reaction system of described pcr amplification is: 10 * damping fluid (contains Mg
2+) 1.0 μ l, the molecule marker primer described in the table 1 of 4pmol/ μ l is to 1 μ l, 2.5mM dNTPs 0.2 μ l, and the Taq enzyme 0.1 μ l of 5U/ μ l, the paddy rice sample gene group template DNA 1 μ l of 10ng/ μ l adds water to 10 μ l; Response procedures is: DNA94 ℃ of pre-sex change is after 5 minutes; 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ are extended exhibition 1 minute, circulate 35 times; Last 72 ℃ were extended 10 minutes.
Beneficial effect
The molecule marking method of the thin rice blast resistant gene of paddy rice provided by the present invention has the following advantages:
(1) thin rice is Taihu Lake basin japonica rice local variety, feature with the high blast resisting of wide spectrum, its main effect disease-resistant gene Pi-bd1 (t) is a new disease-resistant gene, screening obtains closely linked with it molecule marker RM6091 and RM26632, for molecular marker assisted selection breeding and clone Pi-bd1 (t) gene are laid a good foundation.Utilize a pair of arbitrarily and thin closely linked molecule marker of rice blast resistant gene Pi-bd1 (t) of paddy rice of the present invention, carry out the discriminating of blast resistant gene Pi-bd1 (t), efficiency of selection all reaches more than 98.8%, utilizes two pairs of molecule marker selection of primers efficient to reach 99.98%.
(2) accurate by the localized gene locus of molecule marker of the present invention, it is convenient to identify.Because the recombination fraction low (≤1.2%) of these marks and disease-resistant gene Pi-bd1 (t), by detecting these and the closely linked molecule marker of disease-resistant gene Pi-bd1 (t), the existence that can determine blast resistant gene Pi-bd1 (t) whether, the rice blast resistance of prediction rice plant, thereby quick breeding for disease resistance process.
(3) the assistant breeding select target is clear and definite, saves cost.In traditional disease resistant and breeding method, the rice blast resistance of breeding material is carried out phenotypic evaluation, generally at seedling stage or heading stage, it is bigger to be inoculated environmental influence, and the result reliability of phenotypic evaluation is low.Therefore breeding for disease resistance is not only time-consuming, and difficulty is big, the cost height.By detecting blast resistant gene Pi-bd1 (t), can take a sample in seedling stage, extract DNA, utilize above-mentioned mark just can identify the individual plant of blast resisting, eliminate other plant, not only save production cost, control the breeding population scale, and improve the efficiency of selection of blast resisting individuality greatly.
Description of drawings
Fig. 1. the 8% native polyacrylamide gel electrophoresis figure of rice blast resistant gene Pi-bd1 (t) close linkage SSR mark RM6091.
Wherein: M: molecular weight Marker; B: thin rice; S: Suyunuo; F: heterozygous; 1-6: susceptible RIL; 7-13:
Disease-resistant RIL.
Fig. 2. the 8% native polyacrylamide gel electrophoresis figure of rice blast resistant gene Pi-bd1 (t) close linkage SSR mark RM26632.
Wherein: M: molecular weight Marker; B: thin rice; S: Suyunuo; F: heterozygous; 1-5: susceptible RIL; 6-13: disease-resistant RIL.
Embodiment
(1) materials and methods:
Li Peifu etc. utilize the anti-rice blast rice local variety approach rice (♀) and susceptible variety Suyunuo (
) hybridization acquisition F
1, selfing obtains F
2Segregating population, be used for genetic analysis, clearly thin rice is controlled by a pair of main dominant gene of imitating the resistance of " north 1 " fungus strain, further will approach the Japanese rice blast differential varieties hybridization of rice and 12 carries out allelomorphism and measures, the result shows, thin rice is inequipotential to the entrained known disease-resistant gene of the resistant gene of northern 1 fungus strain and 12 differential varieties, is Pi-bd1 (t) (heredity with this unnamed gene, 2007,29 (10): 1249～1255).The present invention adopts single seed descent to make up F on this basis
2: 6Recombinant inbred lines, promptly two parents are hybridized acquisition F
1, selfing obtains F
2, F
2166 individual plants of picked at random, individual plant selfing produce strain system, continuously 5 generations of selfing, finally are built into the F that comprises 166 strains systems
2: 6Recombinant inbred lines.Utilize this colony further Pi-bd1 (t) to be positioned between Sub_clause 11 karyomit(e) RM6091 and the RM26632, and with these two mark close linkages, genetic distance is 1.2CM.
2. spawn culture and inoculation identification method are with reference to (rice in China science, 2007,21:579～584) such as Li Peifu.
3.DNA extracting method: with the DNA of each individual plant of SDS method extraction separation colony.
4. the compact linkage molecule mark is definite:
(1) mark polymorphism screening: the DNA with thin rice of parent and Suyunuo is a template, with the paddy rice SSR mark of announcing on the Gramene website (http://www.gramene.org), through PCR reaction carrying out polymorphism analysis.
(2) the anti-sense pond of polymorphism mark screening: in recombinant inbred lines, select 10 inoculations to be accredited as the tame based material (disease-resistant RIL) of disease-resistant phenotype and the tame based material (susceptible RIL) that 10 inoculations are accredited as susceptible phenotype at random, mix respectively after extracting DNA, form disease-resistant pond and susceptible pond, polymorphism mark is screened itself and the anti-relation between feeling analyzed, if the disease-resistant pond electrophoresis result of a certain mark is consistent with disease-resistant parent, susceptible pond electrophoresis result is consistent with susceptible parent, then illustrate this mark may with the disease-resistant gene close linkage.
(3) checking of compact linkage molecule mark: may in 10 tame based materials that constitute anti-sense pond, verify respectively with the closely linked molecule marker of disease-resistant gene, when if linkage relationship exists really, in all tame based materials, verify again, according to chain polymorphism mark and corresponding anti-sense phenotype, evaluation of markers and intergenic recombination frequency calculate the antagonistic efficiency of selection of mark.
5.PCR reaction system: volume is 10 μ l, and wherein 10 * damping fluid (contains Mg
2+) 1.0 μ l, the molecule marker primer is to (4pmol/ μ l) 1 μ l, 2.5mM dNTPs 0.2 μ l, and Taq enzyme (5U/ μ l) 0.1 μ l, template DNA (10ng/ μ l) 1 μ l adds water to 10 μ l.Response procedures is: DNA94 ℃ of pre-sex change be after 5 minutes, (72 ℃ are extended exhibition 1 minute for 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds) circulation 35 times, and last 72 ℃ were extended 10 minutes.In the enterprising performing PCR amplification of biometre amplification instrument, amplified production carries out electrophoretic separation on 8% non-denaturing polyacrylamide gel (containing 7.6 gram acrylamides and 0.4 gram methylene diacrylamide in the 100ml polyacrylamide solution), on ultraviolet transilluminator, take a picture the record result then.
(2) result and analysis:
Result of study finds that (Fig. 1, Fig. 2), the amplified band size in the parent is as shown in table 2 for SSR mark RM6091, RM26632 and disease-resistant gene Pi-bd1 (t) close linkage.In 166 tame based materials of RIL (in 332 gametes), the efficiency of selection method of calculation are as follows
Occur 2 crossover-type gametes between SSR mark RM6091 and the disease-resistant gene Pi-bd1 (t), recombination fraction only is 1.2%, and the antagonistic efficiency of selection of this mark reaches 98.8%;
Occur 2 crossover-type gametes between SSR mark RM26632 and the disease-resistant gene Pi-bd1 (t), recombination fraction only is 1.2%, and the antagonistic efficiency of selection of this mark reaches 98.8%;
The efficiency of selection of SSR mark RM6091 and the screening of RM26632 double-tagging is 1-1.2%*1.2%=99.98%;
Can identify efficiently that by above-mentioned 2 molecule markers whether blast resistant gene Pi-bd1 (t) exists, single mark efficiency of selection all reaches 98.8%, and the efficiency of selection of double-tagging combination reaches 99.98%.Can be used for the breeding process that molecular marker assisted selection effectively improves the paddy disease-resistant kind, control breeding population scale.
Table 2.
Claims (2)
1. the molecule marking method of the thin rice blast resistant gene Pi-bd1 (t) of paddy rice is characterized in that comprising the steps:
(1) water intaking rice sample extracts paddy rice sample gene group DNA;
(2) utilize any a pair of in the table 1 or two pairs and the thin closely linked molecule marker primer of rice blast resistant gene Pi-bd1 (t) of rice varieties, described paddy rice sample gene group DNA is carried out pcr amplification, pcr amplification product carries out electrophoresis detection on 8% non-denaturing polyacrylamide gel, if amplify the molecule marker fragment of corresponding size, indicate the existence of Pi-bd1 (t) gene;
Table 1
2. the molecule marking method of the thin rice blast resistant gene Pi-bd1 (t) of paddy rice according to claim 1 is characterized in that the reaction system of described pcr amplification is: 10 * contain Mg
2+Damping fluid 1.0 μ l, the described molecule marker primer of 4pmol/ μ l is to 1 μ l, 2.5mM dNTPs 0.2 μ l, the Taq enzyme 0.1 μ l of 5U/ μ l, the paddy rice sample gene group template DNA 1 μ l of 10ng/ μ l adds water to 10 μ l; Response procedures is: 94 ℃ of pre-sex change of DNA are after 5 minutes; 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ are extended exhibition 1 minute, circulate 35 times; Last 72 ℃ were extended 10 minutes.
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102703443A (en) * | 2012-05-23 | 2012-10-03 | 华南农业大学 | Functional specific molecular marker of rice blast resistance gene Pia and method and application thereof |
| CN103205506A (en) * | 2013-05-08 | 2013-07-17 | 辽宁省农业科学院 | Method for detecting rice blast resistant gene pi5 by using coseparation mark pi5-2-4 |
| CN103215369A (en) * | 2013-05-08 | 2013-07-24 | 辽宁省农业科学院 | Co-dominant marker of anti-pyricularia grisea cav. gene pi5 in rice and special primers thereof |
| CN103215368A (en) * | 2013-05-08 | 2013-07-24 | 辽宁省农业科学院 | Method for detecting anti-pyricularia grisea cav. gene pi5 in rice breeding materials by utilizing co-segregation marker pi5-1-2 |
| CN103215370A (en) * | 2013-05-08 | 2013-07-24 | 辽宁省农业科学院 | Method for detecting rice blast-resistant gene pi5 |
| CN103805612A (en) * | 2014-02-08 | 2014-05-21 | 南京农业大学 | Rice gene OsRem1 and application thereof |
| CN105713983A (en) * | 2016-04-21 | 2016-06-29 | 南京农业大学 | Molecular marker closely interlocked with neck blast resistance gene of paddy rice Jiangnan lateness and application thereof |
| CN105779622A (en) * | 2016-04-21 | 2016-07-20 | 南京农业大学 | Molecular marker closely linked with race-specific rice-blast-resistant gene of paddy rice and application of molecular marker |
| CN106555001A (en) * | 2016-11-10 | 2017-04-05 | 福建省农业科学院生物技术研究所 | A kind of molecular labeling of rice blast resistant gene and its application |
| CN106636335A (en) * | 2016-10-09 | 2017-05-10 | 南京农业大学 | Molecular marker method of rice panicle blast resistance gene |
| CN106939346A (en) * | 2017-04-25 | 2017-07-11 | 南京农业大学 | Paddy rice bacterial leaf spot resistant gene xa5 molecular labeling and its application |
| CN106957916A (en) * | 2017-04-25 | 2017-07-18 | 南京农业大学 | Paddy rice bacterial leaf spot resistant gene Xa27 molecular labeling primer and its application |
| CN107130019A (en) * | 2017-04-25 | 2017-09-05 | 南京农业大学 | The molecular labeling primer of the thin spike of rice pest resistant gene of Local Rice Race and its application |
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| CN102703443A (en) * | 2012-05-23 | 2012-10-03 | 华南农业大学 | Functional specific molecular marker of rice blast resistance gene Pia and method and application thereof |
| CN102703443B (en) * | 2012-05-23 | 2014-09-24 | 华南农业大学 | Functional specific molecular marker of rice blast resistance gene Pia and method and application thereof |
| CN103205506A (en) * | 2013-05-08 | 2013-07-17 | 辽宁省农业科学院 | Method for detecting rice blast resistant gene pi5 by using coseparation mark pi5-2-4 |
| CN103215369A (en) * | 2013-05-08 | 2013-07-24 | 辽宁省农业科学院 | Co-dominant marker of anti-pyricularia grisea cav. gene pi5 in rice and special primers thereof |
| CN103215368A (en) * | 2013-05-08 | 2013-07-24 | 辽宁省农业科学院 | Method for detecting anti-pyricularia grisea cav. gene pi5 in rice breeding materials by utilizing co-segregation marker pi5-1-2 |
| CN103215370A (en) * | 2013-05-08 | 2013-07-24 | 辽宁省农业科学院 | Method for detecting rice blast-resistant gene pi5 |
| CN103215369B (en) * | 2013-05-08 | 2014-07-23 | 辽宁省农业科学院 | Co-dominant marker of anti-pyricularia grisea cav. gene pi5 in rice and special primers thereof |
| CN103205506B (en) * | 2013-05-08 | 2014-07-23 | 辽宁省农业科学院 | Method for detecting rice blast resistant gene pi5 by using coseparation mark pi5-2-4 |
| CN103805612A (en) * | 2014-02-08 | 2014-05-21 | 南京农业大学 | Rice gene OsRem1 and application thereof |
| CN105779622A (en) * | 2016-04-21 | 2016-07-20 | 南京农业大学 | Molecular marker closely linked with race-specific rice-blast-resistant gene of paddy rice and application of molecular marker |
| CN105713983A (en) * | 2016-04-21 | 2016-06-29 | 南京农业大学 | Molecular marker closely interlocked with neck blast resistance gene of paddy rice Jiangnan lateness and application thereof |
| CN105713983B (en) * | 2016-04-21 | 2019-03-05 | 南京农业大学 | A kind of molecular labeling and its application with rice Jiangnan evening neck blast resistance gene close linkage |
| CN106636335A (en) * | 2016-10-09 | 2017-05-10 | 南京农业大学 | Molecular marker method of rice panicle blast resistance gene |
| CN106636335B (en) * | 2016-10-09 | 2020-05-08 | 南京农业大学 | Molecular marking method of rice panicle blast resistance gene |
| CN106555001A (en) * | 2016-11-10 | 2017-04-05 | 福建省农业科学院生物技术研究所 | A kind of molecular labeling of rice blast resistant gene and its application |
| CN106555001B (en) * | 2016-11-10 | 2019-07-26 | 福建省农业科学院生物技术研究所 | A kind of molecular labeling of rice blast resistant gene and its application |
| CN106939346A (en) * | 2017-04-25 | 2017-07-11 | 南京农业大学 | Paddy rice bacterial leaf spot resistant gene xa5 molecular labeling and its application |
| CN106957916A (en) * | 2017-04-25 | 2017-07-18 | 南京农业大学 | Paddy rice bacterial leaf spot resistant gene Xa27 molecular labeling primer and its application |
| CN107130019A (en) * | 2017-04-25 | 2017-09-05 | 南京农业大学 | The molecular labeling primer of the thin spike of rice pest resistant gene of Local Rice Race and its application |
| CN107130019B (en) * | 2017-04-25 | 2020-07-17 | 南京农业大学 | Molecular marker primer of rice local variety thin rice panicle blast resistance gene and application thereof |
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