CN111374041A - Method for selecting rice blast resistant rice material with molecular marker assistance - Google Patents

Method for selecting rice blast resistant rice material with molecular marker assistance Download PDF

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CN111374041A
CN111374041A CN201811608691.8A CN201811608691A CN111374041A CN 111374041 A CN111374041 A CN 111374041A CN 201811608691 A CN201811608691 A CN 201811608691A CN 111374041 A CN111374041 A CN 111374041A
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rice
rice blast
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高星
杨坤
姜丽杰
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CHINA NATIONAL JAPONICA RICE RESEARCH AND DEVELOPMENT CENTER
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TIANJIN TIANLONG SEED SCIENCE AND TECHNOLOGY CO LTD
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Abstract

The invention relates to a method for selecting rice blast resistance rice materials with the assistance of molecular markers, which adopts specific molecular markers to select the materials of disease resistance genes with the assistance of the molecular markers, combines the identification of disease resistance in the field, has more definite and efficient breeding targets, and belongs to the field of rice breeding. It includes the following embodiments: 1) hybridizing a resistant parent containing a rice blast resistant gene Pi40 with a susceptible parent Ji Jing 88 to construct a resistant population and identifying and screening progeny populations; 2) DNA extraction, PCR amplification and electrophoresis analysis of the infection-resistant individual plant; 3) the rice variety NG13 containing the rice blast resistance gene Pi40 and the susceptible variety Jijing 88 are planted in Pingyuan city of Jilin province in a rice blast high-incidence area, the rice blast is induced under natural conditions, and the resistance investigation of the leaf blast and the panicle stem blast is respectively carried out in the seedling stage and the mature stage. According to the invention, through the construction of field infection-resistant groups, the molecular markers of the screened rice blast resistance genes Pi40 are utilized, and the resistance identification of rice blast high-incidence areas is combined, so that the novel rice blast-resistant variety can be efficiently and directionally improved and cultivated, and the method has important practical significance for field breeding.

Description

Method for selecting rice blast resistant rice material with molecular marker assistance
Technical Field
The invention relates to a breeding method for selecting a high-rice-blast-resistance material with the assistance of a molecular marker, in particular to a breeding method which adopts a specific molecular marker to select a rice material containing a rice blast resistance gene with the assistance of the molecular marker, combines the identification of field phenotypic characters and disease resistance, has more definite and efficient breeding targets and belongs to the field of rice breeding.
Background
The rice blast is a rice disease caused by rice blast germs and has a very wide distribution range. According to statistics, more than 80 countries occur all over the world, the general popular year can cause 10% -20% of yield reduction, and the serious occurrence reaches 40% -50%. Since the 90 s of the 20 th century, the annual incidence area of rice blast in China is 380 ten thousand hm2Above that, hundreds of millions of kilograms of rice are lost every year. With the simplification and centralization of variety application and the influence of climate, the damage of rice blast is more and more serious. Although people consume huge energy and material investment in pest control, the pest control effect is not satisfactory all the time due to the influence of many factors, and particularly in the years of serious diseases, huge loss is often caused to production and breeding. Taking Jilin province as an example, Jilin province mainly plants a rice variety Jijing 88, and in recent years, due to the disease resistance degradation of the variety, panicle blast and stalk blast occur in a large area, which seriously affects the production of northern japonica rice. The fundamental reason for this phenomenon is that the main cultivar has a narrow spectrum of resistance to rice blast seedlings, and is simple and centralized for widespread planting, so that the cultivar's resistance to rice blast is rapidly reduced once the epidemic races of rice blast germs change. Therefore, the breeding of rice varieties with broad-spectrum resistance is very important.
At present, in the process of breeding rice blast resistant varieties, breeders mainly adopt a pedigree method. The traditional pedigree method selects and breeds preferentially from separated offspring through double basic combinations, the disease-resistant phenotype depends on field natural identification or artificial inoculation identification, the identification difficulty is high, and the accuracy is low. Because the disease-resistant genotype of the parents is not known, a breeder is blindly in parent selection, usually matches groups in a large quantity, selects offspring in a large quantity, and has large workload and low breeding efficiency. The molecular marker is combined with the field disease resistance performance for selection, so that the accuracy and efficiency of breeding resistant varieties can be greatly improved.
The rice blast resistance gene Pi40 is derived from the major disease resistance gene of Australian wild rice (o.australianensis), and has strong resistance to rice blast bacteria from Jilin province in northeast China. Pi40(t) was initially located between DNA markers S2539 and RM3330 on the short arm of chromosome 6 of rice, with genetic distances of 3.8cm and 2.4cm, respectively; 14 BAC/PAC clones were identified in a 1.8Mb contig (conting) on rice Pseudomonas melule 3 by electronic landing (e-plating) technique, and primers designed for 6 NBS-LRR structures were located in PAC clone P0649C 11. The DNA markers P1, P2 are closely linked to Pi 40.
Disclosure of Invention
In order to efficiently select new varieties with high rice blast resistance and specifically select materials containing Pi40 gene offspring, the invention provides an effective breeding method. The DNA markers P1 and P2 are used for molecular marker-assisted selection of the rice blast resistance gene Pi40, and the efficiency of breeding new disease-resistant materials is improved by combining character selection and resistance identification of different separation generations in the field.
The present invention provides the following solutions:
a rice material NG13 containing a rice blast resistance gene Pi40 and a susceptible material Jijing 88 are utilized to construct a susceptible F2 population. Screening individual plants with excellent field phenotypic characters and containing Pi40 by using specific molecular markers P1 and P2, and backcrossing the screened individual plants with the susceptible parent Jijing 88 to obtain a backcross population BC1F1Adding generations in Hainan; sowing BC in Tianjin season1F2The seeds are screened from the rice blast resistance gene Pi40 by the molecular markers P1 and P2 again, meanwhile, disease-resistant single plants are screened by combining field phenotype screening and disease resistance identification, and are backcrossed with susceptible parent Jijing 88 to obtain BC2F1The generation of the population is added in Hainan; sowing BC in Tianjin season2F2Screening the rice blast gene Pi40 by using molecular markers P1 and P2, selfing part of screened disease-resistant single plants for generation addition, and carrying out backcross on the other part of the screened disease-resistant single plants and an susceptible parent Ji-Jing 88; backcross population BC obtained in Tianjin season3F1Generation added in Hainan, Tianjin season for BC3F2The single plant is continuously subjected to disease-resistant gene detection, and the single plant, BC, containing the disease-resistant gene is screened2F3The seeds of (1) continue to be tested for generation addition. At BC2F4Generation and BC3F4And (3) harvesting seeds of single plants during generation, screening disease-resistant genes, carrying out propagation and seed testing on the selected single plants, and selecting a new strain which has similar field phenotype to Jijing 88, contains the rice blast resistance gene Pi40 and has better disease resistance.
A primer set for detecting the presence or absence of the rice blast resistance gene Pi40, such as P1 and P2. The primer sequence of P1 is 5'-CAACAAACGGGTCGACAAAGG-3', and the primer sequence of P2 is 5'-CCCCCAGGTCGTGATACCTTC-3'.
A method for detecting whether a rice blast resistance gene Pi40 exists in rice DNA takes sequences shown in P1 and P2 as primers to amplify the DNA to be detected. The band containing NG13 in the amplified product is the rice blast resistance gene Pi40, as shown in the 1 st hole of FIG. 1.
A method for selectively breeding a rice blast resistant material comprises the steps of taking a rice material containing a rice blast resistant gene Pi40 as a parent, hybridizing the rice material with a susceptible rice variety, separating individual plants of a colony of F2 as a filial generation, extracting genome DNA, carrying out PCR amplification by taking sequences shown in P1 and P2 as primers, and screening individual plants which contain the rice blast resistant gene Pi40 and have better disease resistance by combining field disease resistance and phenotype identification.
The primer pair preferentially marks and identifies the DNA extracted from the rice seedling stage, detects whether the DNA carries the rice blast resistance gene Pi40, verifies the identification result of the primer by combining with the field disease resistance detection, and has higher accuracy. The primer pair can be used as a molecular marker of Pi40 gene in rice blast resistance breeding.
Drawings
FIG. 1 is an agarose gel electrophoresis chart of the amplified products of the segregation populations of the resistant parent NG13 and the susceptible variety Jijing 88 of the rice blast resistance gene Pi40 and progeny of the molecular markers P1 and P2:
m: 2000bp molecular weight marker (fragment length 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp respectively)
1; NG 132; jijing 883, 5, 7, 9, 10 and 11 are susceptible single-strain types; 4, 6, 8, 12 are disease-resistant single plant banding patterns
Detailed Description
Example 1: construction of resistant population by crossing resistant parent containing rice blast resistance gene Pi40 with susceptible parent Jijing 88 and identification and screening of progeny population
The resistant parent NG13 containing the rice blast resistance gene Pi40 is hybridized with the susceptible parent Jijing 88 and then self-bredThen selfing the F2 generation to perform character screening and rice blast resistance gene Pi40 screening, and selecting 50F 2 single plants which are similar to Jijing 88 in plant height, growth period, high yield and the like and contain a resistance gene Pi 40; then backcrossing with susceptible parent Ji Jing 88 for the first time to obtain BC1F2Then, the method is carried out with character screening and screening of disease-resistant gene Pi40, and BC which has excellent characters and contains disease-resistant gene Pi40 is selected1F250 single plants; then carrying out second backcross with susceptible parent Ji Jing 88, and carrying out selfing to obtain BC2F2Then, the method selects the characters and the disease-resistant gene Pi40, selects the BC with excellent characters and containing the disease-resistant gene2F2One part of the single plant is self-bred and added with generation, the other part is backcrossed with susceptible parent Ji Jing 88 for the third time, and the BC is obtained by self-breeding3F2Then field phenotype screening and screening of disease-resistant gene Pi40 are carried out to select BC with excellent properties and containing disease-resistant gene3F2Selfing the single plant; BC2F2Progeny from selfing and BC3F2And (4) screening field phenotypic characters and identifying a disease-resistant gene Pi40 in selfed offspring. To BC2F4And BC3F4Selecting a strain with excellent phenotype and containing disease-resistant genes, carrying out local propagation, yield measurement, seed test and rice blast resistance overall identification, and breeding a new rice blast-resistant strain according to the whole breeding process.
Example 2: DNA extraction, PCR amplification and electrophoresis analysis of anti-influenza individual plant
DNA extraction (CTAB method)
0.1-0.2 g of rice leaves were weighed, cut to a length of 1cm, and placed in a 800. mu.l centrifuge tube containing an extract (1M Tris pH8.0, 100 mM: 5M NaCl, 1.0M EDTA, 20 mM: 10% CTAB, 2.0%) preheated at 65 ℃. Placing the tissue grinding instrument in a tissue grinder with the frequency of 57.0HZ, the rotating speed of 1710N and the vibration of 200S. Water bath at 65 deg.C for 30min, and gently shaking every 10 min. An equal volume of phenol chloroform/isoamyl alcohol (25: 24: 1) was added and gently shaken to form an emulsion. Centrifuging at 12000r/min for 10min at room temperature (the temperature is not lower than 15 deg.C), and collecting supernatant. Carefully extracting 450 μ l of supernatant, adding 2 times volume of precooled anhydrous ethanol (or isovolumetric isoamyl alcohol), standing at 20 deg.C for more than 20min, centrifuging at 5000r/min for 5min, and collecting precipitate. Washing the precipitate with 70% ethanol for 1-2 times. And opening the tube cover, putting the tube cover in a clean place, and airing, wherein the milky white DNA precipitate is transparent. Finally, 100. mu.l of TE (Tris-HCl110mM, pH 8.0; EDTA1mM, pH8.0) was added to dissolve the precipitate, and the precipitate was diluted to 100. mu.g/ml and stored at-20 ℃ for further use.
Secondly, PCR amplification:
PCR reaction System 2 × Taq PCR Master Mix 10. mu.l, 10. mu.M primers 0.5. mu.l each, 100ug/ml template DNA 1. mu.l, ddH2O8μl。
PCR reaction procedure: pre-denaturation at 95 ℃ for 3 min; circulating for 35 times at 95 deg.C for 30s, 65 deg.C for 30s, and 72 deg.C for 1 min; extending for 10min at 72 ℃, and storing at 4 ℃.
Third, PCR detection
And (3) detecting the result by agarose gel electrophoresis, adding 6 mu l of PCR product, staining by gold view, and taking a picture by using a gel imaging system.
Example 3: the disease-resistant variety NG13, the susceptible variety Ji Jing 88 and the susceptible group single plants containing the rice blast resistance gene Pi40 are planted in the pine city of Jilin province in the rice blast high-incidence area, the rice blast is induced under natural conditions, the resistance investigation of the leaf blast and the panicle stem blast is respectively carried out in the seedling stage and the mature stage, and the unified standard is used for evaluation.
1. Field identification of leaf blast
Classical record of the leaf blast:
grade 0 disease-free … … … … … … … … … … … … … … … … … … … … … … … … … High Resistance (HR)
Grade 1 lesion is needle-like large and small brown point … … … … … … … … … … … … … … … … … anti (R)
Grade 2 slightly brown point … … … … … … … … … … … … … … … … … … … … … … … anti (R)
Grey spots ranging from circular to elliptical in grade 3, brown at the edge, Moderate (MR) in the spot diameter of about 1-2 mm … …
Grade 4 typical spindle-shaped lesions, 1-2 cm long, usually confined between the two veins, with an area of injury not exceeding 2% of the area of the leaves in … … feeling (MS)
Grade 5 typical spindle-shaped lesions, which are perceived as being in … … … … … … … (MS) with lesion area not exceeding 10% of leaf area
Typical spindle-shaped disease spots of grade 6, which are … … … … … … … … … … … -sense (S) with the area of injury of 10.1-25%
Typical spindle-shaped disease spots of grade 7, which are … … … … … … … … … … … -sense (S) with an area of injury of 25.1-50%
Typical spindle-shaped disease spots of grade 8, High Sensitivity (HS) with an affected area of 50.1-75% … … … … … … … … … … …
High-sensitivity (HS) … … … … … … … … … … … … … … … … … … … … … dead blade of 9-grade
After the field natural induction, the epidemic rice blast disease leaf blast in the pine original area of Jilin province is identified. The results show that the rice blast resistance gene Pi40 donor parent NG13 and the disease-resistant individuals of No. 4, No. 6, No. 8 and No. 12 show disease resistance (R), and when the landholder susceptible variety Jijing 88 is highly susceptible, the individuals of No. 3, No. 5, No. 7, No. 9, No. 10 and No. 11 show susceptibility (S).
2. Neck blast field identification
Evaluation and grading standard for panicle blast disease spike rate
Grade 0 disease-free … … … … … … … … … … … … … … … … … … … … … … High Resistance (HR)
Grade 1 ear rate is less than 1 to 5% … … … … … … … … … … … … … … … … Fang (R)
Grade 3 spike rate of resistance (MR) in 5.1-10% … … … … … … … … … … … … … … …
Grade 5 ear rate of disease in 10.1-25% … … … … … … … … … … … … … … … sense (MS)
Grade 7 ear disease rate of 25 ~ 50 … … … … … … … … … … … … … … … … … Gai (S)
Grade 9 ear rate of disease 50.1 ~ 100% … … … … … … … … … … … … … … Gao gan (HS)
After the natural induction in the field, the neck blast of the rice blast disease epidemic in the pine region of Jilin province is identified. The results show that the rice blast resistance gene Pi40 donor parent NG13 and the disease-resistant individuals of No. 4, No. 6, No. 8 and No. 12 show disease resistance (R), and when the landholder susceptible variety Jijing 88 is highly susceptible, the individuals of No. 3, No. 5, No. 7, No. 9, No. 10 and No. 11 show susceptibility (S).
In the seedling stage, genomic DNA was extracted as described in example 2, and PCR amplification was performed using P1 and P2 as primers, the results of which are shown in FIG. 1: the rice blast resistance gene Pi40 donor parent NG13 and the banding pattern of the disease-resistant single plant contain two bands of 200bp and 120bp, and the susceptible parent Ji Jing 88 and the banding pattern of the susceptible single plant contain two bands of 250bp and 200 bp.
Thus, the rice blast resistance gene Pi40 can be identified well by using the molecular markers P1 and P2, the field performance resistance of the material containing the Pi40 gene is good, and the field performance susceptibility of the material without the Pi40 gene is good.

Claims (2)

1. A method for selecting rice blast resistant rice materials with the assistance of molecular markers is characterized by comprising the following steps:
the rice material NG13 containing the rice blast resistance gene Pi40 is used as a female parent, the conventional japonica rice variety Jijing 88 susceptible to rice blast is used as a male parent for hybridization, the offspring is directionally selected, the single progeny plant with agronomic characters biased towards japonica rice variety Jijing 88 is selected, the molecular marker is used for detection, and meanwhile, the field disease resistance identification is carried out in a rice blast high-incidence area, so that the new variety of the Jijing 88 with high rice blast resistance is bred.
2. The breeding method as claimed in claim 1, wherein the directional selection in step (1) is carried out by crossing rice material NG13 containing rice blast resistance gene Pi40 with susceptible material Jijing 88, selfing, carrying out trait selection and single plant detection containing rice blast resistance gene Pi40 in F2 generation by selfing. Selecting F2 single plant with excellent character and containing disease-resistant gene, backcrossing with susceptible japonica rice variety Jijing 88 for one time, selfing to obtain BC1F2Then, trait selection and single plant detection containing the rice blast resistance gene Pi40 were performed. Selection of BC having excellent traits and containing disease-resistant gene Pi401F2The single plant is backcrossed with the susceptible japonica rice variety Jijing 88 for the second time, and the BC is obtained by selfing2F2Then, character selection and single rice blast resistance gene Pi40 are carried outAnd (5) detecting strains. Selection of disease-resistant Gene-containing BC having Excellent Properties2F2One part of the single plant is self-bred and added with generation, the other part is backcrossed with the susceptible japonica rice variety Jijing 88 for three times, and the BC is obtained by self-breeding3F2Then, trait selection and single plant detection containing the rice blast resistance gene Pi40 were performed. Selection of disease-resistant Gene-containing BC having Excellent Properties3F2And (4) selfing the single plants. BC2F2Progeny from selfing and BC3F2In the selfing progeny, the field phenotypic character selection and the identification of the disease-resistant gene Pi40 are carried out, and BC is selected2F4And BC3F4The strain containing disease-resistant gene with excellent phenotype is locally propagated, tested for production, tested for species and identified for rice blast resistance.
CN201811608691.8A 2018-12-27 2018-12-27 Method for selecting rice blast resistant rice material with molecular marker assistance Pending CN111374041A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114410731A (en) * 2021-12-02 2022-04-29 吉林省农业科学院 Grate screen for screening rice blast resistant materials and application thereof
CN117305506A (en) * 2023-11-07 2023-12-29 四川省农业科学院植物保护研究所 Accurate rice false smut resistance gene identification method

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114410731A (en) * 2021-12-02 2022-04-29 吉林省农业科学院 Grate screen for screening rice blast resistant materials and application thereof
CN114410731B (en) * 2021-12-02 2024-02-27 吉林省农业科学院 Grate screen for screening rice blast resistance material and application thereof
CN117305506A (en) * 2023-11-07 2023-12-29 四川省农业科学院植物保护研究所 Accurate rice false smut resistance gene identification method
CN117305506B (en) * 2023-11-07 2024-05-28 四川省农业科学院植物保护研究所 Accurate rice false smut resistance gene identification method

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