CN104429928A - Method for improving identification accuracy of rice blast resistance - Google Patents
Method for improving identification accuracy of rice blast resistance Download PDFInfo
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- CN104429928A CN104429928A CN201410699984.7A CN201410699984A CN104429928A CN 104429928 A CN104429928 A CN 104429928A CN 201410699984 A CN201410699984 A CN 201410699984A CN 104429928 A CN104429928 A CN 104429928A
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Abstract
The invention discloses a method for improving the identification accuracy of rice blast resistance. According to the method, the identification accuracy of the rice blast resistance is quickly improved by utilizing false positive rate, false negative rate, a positive predictive value and a negative predictive value, and combining a field identification result. The method has the benefits that human factors are reduced, and the identification efficiency and accuracy are improved.
Description
Technical field
The present invention relates to a kind of blast resistance identification method, particularly a kind of method improving blast resistance identification accuracy.
Background technology
Paddy rice is one of most important cereal crops in the whole world.China produces the maximum country of paddy in the world per year, and the whole nation has the population of more than 60% to take rice as staple food.Current rice blast is the major obstacle of Rice Production, the Rice Yield Loss Caused that the whole world is caused by rice blast is every year enough to support more than 6,000 ten thousand people, cause the loss of nearly 5,000,000,000 dollars, according to estimates, the global paddy caused by rice blast between 16 years of 1975 ~ nineteen ninety loses up to 1.57 hundred million t, annual 1000 ten thousand about t.Since the nineties, China's rice blast year occurring area all at 3,800,000 hm
2above, annual loss paddy reaches several hundred million kilograms.In the Hybrid Rice Combinations of nearly 5 years southern each province and country's authorization, account for 87% to the paramount sense of rice blast sense, resistant variety is few.Even the new rice varieties with blast resisting be bred as is after continuous 3-5 generation plantation, resistance against diseases often shows as decline, and disease is in the trend constantly increased the weight of.
Traditional blast resistance identification depends on plant phenotype and observes, and requires rich experience and longer time, and is subject to the restriction of rice blast onset condition, and lacks the effective method for tracing of enantiopathy gene, is difficult to realize precise Identification.Along with the development of rice genome and transgenic technology, for blast resistance identification opens new path.Rice blast is caused by fungi sac fungi Magnaporthe grisea (Hebert) Barr. (Invisible element Pyriculariag griseaSacc.), and germ is easy to morph, and there is different biological strains.China sets up rice blast biological strain Combined Trials group for 1976, proposes 7 unified differential varieties of the whole nation and authentication method, in the control of rice blast, serves positive effect.But the rice blast differential variety of China selects by rule of thumb artificially according to its response type to the some fungus strains of Pyricularia oryzae, subjectivity is large.China once used this cover differential variety and authentication method to be engaged in the Resistance Identification work of the monitoring of rice blast biological strain and rice varieties.Because the disease-resistant gene situation awareness had differential variety obtains seldom, be difficult to the essential difference disclosing rice blast biological strain from genetic aspect, in actual application, there is distinguishing ability not strong, the limitation that accuracy is not high.In recent years, the near-isogenic line of blast resisting and the incubation of Monogenic lines, for studying the toxicity composition of Pyricularia oryzae in certain area, the regionality of the structure and distribution and disease-resistant gene of verifying virulence gene utilizes and provides technical support.Meanwhile, along with molecular biological development, the molecular marking technique be based upon on DNA polymorphism basis is then the genetic lineage of research Pyricularia oryzae, the disease-resistant gene of qualification disease-resistant variety becomes a reality.Utilize the change of research in new high-tech Chongqing City rice blast pathogen biological strain, distribution, toxicity composition and genetic lineage, improve the accuracy of rice blast monitoring qualification.
Blast resistance identification adopts artificial infection idenfication always, is still major diagnostic means now.But conventional identification technology is subject to the impact of environmental condition and pathogenic factors, and appraiser requires rich experience, be difficult to the accuracy ensureing qualification.Adopt Molecular Identification to identify with artificial Field inoculation the method combined, improve qualification accuracy and efficiency.So far, this technology has no report.For many years, blast resistance identification is all artificial infection, according to the artificial subjective judgement of phenotype, can not carry out substance qualification from molecule angle.
Summary of the invention
The object of the invention is to solve the problem, devising a kind of method improving blast resistance identification accuracy.
Realizing above-mentioned purpose technical scheme of the present invention is that a kind of method improving blast resistance identification accuracy, the method comprises the steps:
(1), to educate 69-1 for maternal and Lac23 for male parent is hybridized, F is obtained
1for hybrid seed 500, to F
1carry out cultivation for seed and obtain F
2for plant population, F
2press the individual plant area plantation of 16.5cm × 26.4cm for plant population, every 12 strains are a line, often the wide 67cm in aisle in the ranks, and the mode then added for selfing by simple grain colony is bred to F
8in generation, obtain recombinant inbred lines 297 strain; (2), F is gathered in tillering stage
8generation and parent's blade, adopt modified CTAB method to extract STb gene.Utilize the SSR marker near Pi-1 gene, to educate the DNA of 69-1 and Lac23 for template, between parents, filter out difference mark 5, at F
8consistent individual plant is marked with Lac23 for selecting in RIL; (3), to F
8for colony's rice blast field test, sick garden to be combined with natural induction in fields by human assistance inoculation and carries out the Resistance Identification of leaf pest, panicle blast, and rice blast field grade scale investigates by the rice blast in National Standard of the People's Republic of China (GB/T1579-1995) and the standard of observing and predicting is carried out; (4), utilize Molecular Identification data, calculate false positive rate and false negative rate, positive predictive value and negative predictive value.Positive predictive value refers to the disease-resistant material ratios in field contained in the positive band that molecule experiments is checked out, and for RM224, ratio is 12/31.Negative predictive value refers in molecule experiments the ratio checking out material not disease-resistant containing removing field in negative band, for RM224, ratio is (266-52)/266, then mark diagnostic test evaluation value is calculated by positive predictive value × negative predictive value/false positive rate × false negative rate, relatively 5 priorities be marked in blast resistance identification, in conjunction with the accuracy of field test result verification Molecular Identification result.
Wherein, described to F
1carry out cultivation for seed and obtain F
2the F to obtaining is referred to for plant population
1hybrid seed carries out miscegenation.Described simple grain colony adds and refers at F for selfing
2for Stochastic choice in plant population 500 strain, 1 seed is only received in every strain, F
3again carry out miscegenation and obtain F
4for plant population, F
4receive 1 seed for the every strain of plant population, then choose a F
4for seed self propagated to F
8generation.Shown in the described primer sequence for Molecular Identification table composed as follows:
。
The method of the raising blast resistance identification accuracy utilizing technical scheme of the present invention to make, adopt rice blast resistance molecular engineering, Resistance Identification result is disclosed from heredity, achieve Traditional Man inoculated identification technology and molecular biotechnology organically combines, reduce human factor, improve determination rates and accuracy.
Accompanying drawing explanation
Fig. 1 is Molecular Identification of the present invention and Field inoculation qualification result analytical table;
Fig. 2 is the contrast table of testing result of the present invention and field result.
Embodiment
Below the present invention is specifically described, first selects Chongqing City's local supplies material to educate 69-1 for maternal, with from Libya, carry wide spectrum, the rice material LAC23 of dominant blast resistant gene Pi-1 is that male parent carries out hybridization acquisition F
1for seed, F
1for miscegenation, at F
2stochastic choice 500 strain in colony, 1 seed is only received in every strain, F
3miscegenation again, 1 seed is received in every strain, passes self propagated to F with this
8, obtain recombinant inbred lines 297 strain.F is gathered in tillering stage
8and parent's blade, adopt modified CTAB method to extract STb gene (Song Min etc., 2001).Utilize the SSR marker near Pi-1 gene, to educate the DNA of 69-1 and Lac23 for template, between parents, filter out difference mark 5, at F
8select in RIL to mark consistent individual plant with Lac23, utilize Molecular Identification data, calculate false positive rate and false negative rate is low, positive predictive value and negative predictive value.Be mark diagnostic evaluation of estimate with positive predictive value × negative predictive value/false positive rate × false negative rate, relatively 5 priorities be marked in blast resistance identification, the preferential use order detected in this experiment containing Pi-1 genetic material is RM6293 > RM254 > RM224 > RM2136 > RM6094, the results are shown in Table 1 and table 2.F
8colony to be combined with natural induction in fields by human assistance inoculation and carries out the resistance of leaf pest, panicle blast, and qualification result as shown in Figure 2.
In specific operation process, should be noted that following content:
1, parent educates 69-1 and LAC23
Educating 69-1 is a part Chongqing Germplasm Resources, has been submitted to Institute of Crop Science, Chinese Academy of Agricultural Science by breeding qualification and has carried out arranging and cataloging, and enters the long-term storehouse of country and preserve.The feature of this resource: plant height 118cm, strain shape is compact, the thick shape of cane, and grain is yellow; long grain type, few awns, bran point is colourless, about the 150 days time of infertility; than bright extensive 63 short 1-2 days, point evil power is strong, spike length 26.3; fringe grain 168.8, real grain 145.6, ripening rate 86.3%; thousand kernel weight 28.9 grams, the long 9.6mm of grain, the wide 3.1mm of grain; aspect ratio 3.2, Coarse Rice Rate 83.5%, polished rice rate 74.7%.Blast resisting donor parents LAC23, from Libya, carries Pi-1 gene.
2, Molecular Identification, extract LAC23 and educate 69-1 and recombinant inbred lines 297 pnca gene group STb gene, to show the molecular labeling RM224 of difference in parents, RM254, RM2136 with the wide spectrum anti-rice blast tight chain lock of gene Pi-1, RM6094, RM6293 is primer, is that template carries out pcr amplification with parent dna, is that pcr amplification is carried out in contrast with LAC23, consistent with male parent LAC23 band is designated as 1, and consistent with maternal band is designated as 0.Amplified reaction mixeding liquid volume is 20 μ l, and wherein 10 × PCR buffer solution is (containing Mg2+, Buffer:100mMTris.Cl pH 9.0; 500mM KCl; 18mM MgCl
2; 1%Triton X-100) 2.0 μ l, 2.5mmo l.L-1dNTP 1.5 μ l, 5U. μ l-1TaqDNA polymerase 0.5 μ l, 2 μm of o l.L-1SSR primer 2 .0 μ l, 20ng. μ l-1 template DNA 2.0 μ l, ddH
2o 12.0 μ l.The response parameter of PCR is: 94 DEG C of 5min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, and totally 35 circulations, then 72 DEG C of 10min, take out at being placed on 4 DEG C for subsequent use after temperature is down to 10 DEG C.After pcr amplification, add 2 μ l bromjophenol blue indicator, after mixing, with being added with 3% Ago-Gel of ethidium bromide in 0.5 × TBE (0.5 × TBE:1.045mol/Tris boric acid; 0.001mol/L EDTA) constant voltage 120V-150V electrophoresis 2h-3h in buffer solution, by gel imaging system scanning record electrophoresis result.
3, blast resisting Field inoculation qualification, carries out artificial spray inoculation by the mixing spore liquid of isolated strains to bringing out kind rice shoot, and by Pathogen culture Ji Sashi in ridge surrounding.Beating railway carriage or compartment with bringing out kind (Lijiang xintuanheigu and territory excellent 329) during transplanting, bringing out kind anti-spectrum of planting in the ranks and measuring the sick rice seedling after investigation, middle plant F
8colony.Only control worm at rice growing season and do not control rice blast.In the rice tillering later stage, sick garden leaf pest enter popular Sheng and send out the phase and investigate, and after one week, check once; Stage of yellow ripeness panicle blast stable disease " Invest, Then Investigate " neck pest.Comprehensive resistance reach more than 3 grades for disease-resistant individual plant.
4, Molecular Identification and Field inoculation qualification result are analyzed, and statistics molecule appraising datum, calculates false positive rate and false negative rate is low, positive predictive value and negative predictive value.Be mark diagnostic evaluation of estimate with positive predictive value × negative predictive value/false positive rate × false negative rate.According to the size of mark diagnostic evaluation of estimate, relatively 5 priorities be marked in blast resistance identification, the preferential use order detected in this experiment containing Pi-1 genetic material is RM6293 > RM254 > RM224 > RM2136 > RM6094, in the detection respectively to 297 materials of the 5 pairs of SSR primers, occur that the material identical with maternal band is nearly all not disease-resistant, accuracy is all greater than 80%, the negative predictive value of RM2136 up to 91.56%, as shown in Figure 1.As can be seen from Table 2, positive disease-resistant rate is all greater than negative disease-resistant rate, in 41 materials that primer RM6293 increases out identical with male parent band, wherein have 23 disease-resistant, account for 56.10%, disease-resistant ratio is the highest.RM2139 is minimum, filters out the material identical with male parent band 131, wherein only have 50 disease-resistant, account for 38.16%.The material that 5 pairs of primer amplifications band is out all identical with male parent has 1, and is disease-resistant material.The material that amplification band is out all identical with female parent has 105, all shows as susceptible.
Technique scheme only embodies the optimal technical scheme of technical solution of the present invention, and those skilled in the art all embody principle of the present invention to some variations that wherein some part may be made, and belong within protection scope of the present invention.
Claims (4)
1. improve a method for blast resistance identification accuracy, it is characterized in that, the method comprises the steps:
(1), to educate 69-1 for maternal and Lac23 for male parent is hybridized, F is obtained
1for hybrid seed 500, to F
1carry out cultivation for seed and obtain F
2for plant population, F
2press the individual plant area plantation of 16.5cm × 26.4cm for plant population, every 12 strains are a line, often the wide 67cm in aisle in the ranks, and the mode then added for selfing by simple grain colony is bred to F
8in generation, obtain recombinant inbred lines 297 strain;
(2), F is gathered in tillering stage
8generation and parent's blade, adopt modified CTAB method to extract STb gene.Utilize the SSR marker near Pi-1 gene, to educate the DNA of 69-1 and Lac23 for template, between parents, filter out difference mark 5, at F
8consistent individual plant is marked with Lac23 for selecting in RIL;
(3), to F
8for colony's rice blast field test, sick garden to be combined with natural induction in fields by human assistance inoculation and carries out the Resistance Identification of leaf pest, panicle blast, and rice blast field grade scale investigates by the rice blast in National Standard of the People's Republic of China (GB/T1579-1995) and the standard of observing and predicting is carried out;
(4) Molecular Identification data, are utilized, calculate false positive rate and false negative rate, positive predictive value and negative predictive value, then calculate mark diagnostic test evaluation value by positive predictive value × negative predictive value/false positive rate × false negative rate, compare 5 priorities be marked in blast resistance identification.In conjunction with the accuracy of field test result verification Molecular Identification result.
2. the method for raising blast resistance identification accuracy according to claim 1, is characterized in that, described to F
1carry out cultivation for seed and obtain F
2the F to obtaining is referred to for plant population
1hybrid seed carries out miscegenation.
3. the method for raising blast resistance identification accuracy according to claim 1, is characterized in that, described simple grain colony adds and refers at F for selfing
2for Stochastic choice in plant population 500 strain, 1 seed is only received in every strain, F
3again carry out miscegenation and obtain F
4for plant population, F
4receive 1 seed for the every strain of plant population, then choose a F
4for seed self propagated to F
8generation.
4. the method for raising blast resistance identification accuracy according to claim 1, is characterized in that, shown in the described primer sequence for Molecular Identification table composed as follows:
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106376455A (en) * | 2016-01-16 | 2017-02-08 | 魏伟 | Method for breeding disease-resistant rice variety |
| CN106957890A (en) * | 2017-03-22 | 2017-07-18 | 恩施土家族苗族自治州农业科学院 | A kind of method for improving rice varieties fringe pest indoors artificial inoculated identification accuracy |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101485283A (en) * | 2008-12-29 | 2009-07-22 | 重庆市农业科学院生物技术研究中心 | Method for improving selection efficiency in hybrid rice breeding |
| CN102703499A (en) * | 2012-06-27 | 2012-10-03 | 浙江省农业科学院 | Method for converting disease-resistance genes of rice and obtaining transgenic descendants without selective markers |
-
2014
- 2014-11-27 CN CN201410699984.7A patent/CN104429928A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101485283A (en) * | 2008-12-29 | 2009-07-22 | 重庆市农业科学院生物技术研究中心 | Method for improving selection efficiency in hybrid rice breeding |
| CN102703499A (en) * | 2012-06-27 | 2012-10-03 | 浙江省农业科学院 | Method for converting disease-resistance genes of rice and obtaining transgenic descendants without selective markers |
Non-Patent Citations (1)
| Title |
|---|
| 姚南等: "利用 Pi-1 基因邻近SSR标记鉴定稻瘟病抗性", 《分子植物育种》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106376455A (en) * | 2016-01-16 | 2017-02-08 | 魏伟 | Method for breeding disease-resistant rice variety |
| CN106957890A (en) * | 2017-03-22 | 2017-07-18 | 恩施土家族苗族自治州农业科学院 | A kind of method for improving rice varieties fringe pest indoors artificial inoculated identification accuracy |
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