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Method for detecting anti-pyricularia grisea cav. gene pi5 in rice breeding materials by utilizing co-segregation marker pi5-1-2

A rice blast resistance gene and rice blast resistance technology, applied in the field of molecular biology, can solve the problems of low accuracy, heavy workload, blindness, etc.

Inactive Publication Date: 2013-07-24
LIAONING ACAD OF AGRI SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional pedigree method selects the best breeding varieties from the separated offspring through the combination of two parents. The blast resistance phenotype depends on natural identification in the field or artificial inoculation identification, which is difficult to identify and has low accuracy.
Because they do not know the blast resistance genotype of the parents, the breeders are very blind in the selection of the parents, usually mass matching, massive selection of offspring, heavy workload and low breeding efficiency

Method used

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  • Method for detecting anti-pyricularia grisea cav. gene pi5 in rice breeding materials by utilizing co-segregation marker pi5-1-2
  • Method for detecting anti-pyricularia grisea cav. gene pi5 in rice breeding materials by utilizing co-segregation marker pi5-1-2
  • Method for detecting anti-pyricularia grisea cav. gene pi5 in rice breeding materials by utilizing co-segregation marker pi5-1-2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: DNA extraction, PCR amplification and electrophoresis analysis

[0020] 1. DNA extraction (CTAB method):

[0021] Weigh 0.1-0.2 g of rice leaves and cut them into 1 cm lengths, grind them in liquid nitrogen, and after the liquid nitrogen has evaporated (note: do not let the samples melt), quickly transfer them to extracts containing 65°C preheated (1M Tris pH8.0, 100mM; 5M NaCl, 1.0M; 0.5M EDTA, 20mM; 10% CTAB, 2.0%) in a 400μL centrifuge tube. Shake gently.

[0022] 60~65℃ water bath for 30~60 minutes, shake gently every 10 minutes evenly.

[0023] Add an equal volume of chloroform / isoamyl alcohol (24:1), and shake gently to form an emulsion.

[0024] At room temperature (the temperature is not lower than 15° C.), centrifuge at 12000 r / min for 5 minutes, and take the supernatant.

[0025] Carefully extract 200 μL of the supernatant with a large-hole tip, add pre-cooled 2 times the volume of absolute ethanol (or an equal volume of isoamyl alcohol), place ...

Embodiment 2

[0036] Example 2: Detection of pi5-1-2 markers in pi5 donor varieties and monogenic lines.

[0037] Using rice blast resistance gene Pi5 donor variety RIL260, pi5 monogenic line and 5 highly susceptible rice varieties (Lijiang Xintuan Heigu; Liaoyan 241; Yanfeng 47; Liaoxing 1; Nipponbare) as test materials, planted in In the greenhouse, the blast fungus ZA9, ZA33, ZB1 and ZB17 popular in the Liaoning area were transplanted on the oatmeal tomato juice agar medium, and cultured at 25-27°C for 5-7 days. After wiping off the aerial hyphae with cotton swabs and cultivating them with moisture, the blast fungus will produce a large number of spores. When the rice seedlings of 7 varieties grow to 5 leaves and 1 heart, the conidia of each bacterial strain cultivated above are washed with 120ml sterile water respectively, filtered with double-layer gauze and packed into a triangular flask, and the spore suspension (concentration There are about 20 spores under the microscope field of ...

Embodiment 3

[0053] Example 3: Using the molecular marker pi5-1-2 to detect whether the breeding parent material contains the rice blast resistance gene pi5

[0054] Using rice blast resistance gene Pi5 donor varieties RIL260, pi5 single-gene lines (introduced from IRRI) and 16 rice varieties as test materials, they were normally planted in the field, and DNA was extracted according to the method described in Example 1 at the seedling stage. NO: 1 and SEQ ID NO: 2 are primers for PCR amplification, and the amplification results of 18 varieties are as follows figure 2 As shown, in addition to the rice blast resistance gene Pi5 donor variety RIL260 and the pi5 single-gene line (introduced from IRRI), the 13th variety Liaonong 979 also amplified specific products consistent with the donor variety and the single-gene line. Sequencing results showed that the sequence of the amplified product sequence shown in SEQ ID NO: 3 was consistent. The results of inoculation identification showed that L...

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Abstract

The invention belongs to the field of molecular biology and in particular relates to a molecular marker for detecting an anti-pyricularia grisea cav. gene in rice and special primers thereof. The molecular marker is a dominant molecular marker pi5-1-2 of the anti-pyricularia grisea cav. gene pi5. The molecular marker uses a primer pair SEQ ID NO:1 and SEQ ID NO:2 to amplify a nucleotide sequence from the total DNA (deoxyribonucleic acid) of rice, and can be applied to molecular marker-assisted selection of pi5 in rice pyricularia grisea cav. resistance breeding to improve the disease-resistant variety breeding efficiency and reduce the workload of field identification.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to detecting blast resistance gene pi5 in rice breeding materials by using co-segregation marker pi5-1-2. Background technique [0002] Rice blast (Pyricularia grisea cav.) is a rice disease caused by Magnaporthe oryzae (Anamorphic: Pyricularia), and its distribution is very wide. According to statistics, it occurs in more than 80 countries in the world, and it can cause a 10%-20% reduction in production in general epidemic years, and it can reach 40%-50% in severe cases. Since the 1990s, the annual occurrence area of ​​rice blast in my country has been more than 3.8 million hm2, and the annual loss of rice has reached hundreds of millions of kilograms. With the simplification and centralization of varieties and the impact of climate, the harm of rice blast is becoming more and more serious. Although people have spent a lot of energy and material input on disease prevention and co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11A01H1/04
Inventor 白春明郑文静陆晓春赵家铭张丽霞
Owner LIAONING ACAD OF AGRI SCI
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