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Molecular breeding method for new rice variety carrying gene Pi65(t) with resistance to rice blast

A rice blast resistance gene and rice technology, applied in the field of molecular biology, can solve the problems of narrow resistance spectrum and decreased resistance of varieties to rice blast.

Active Publication Date: 2015-07-15
LIAONING ACAD OF AGRI SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because rice blast is a race-specific disease, the interaction between pathogenic bacteria and rice follows the "gene-to-gene" model. Generally, once the endemic race of M. oryzae changes, the variety's resistance to blast will decline rapidly

Method used

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  • Molecular breeding method for new rice variety carrying gene Pi65(t) with resistance to rice blast
  • Molecular breeding method for new rice variety carrying gene Pi65(t) with resistance to rice blast
  • Molecular breeding method for new rice variety carrying gene Pi65(t) with resistance to rice blast

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 pH 8.0, 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: Polymorphism analysis between disease-resistant genes in donor varieties and alleles in susceptible varieties

[0037] The rice blast resistance gene Pi65(t) donor variety Gangyu 129 and 8 susceptible rice varieties (Liaoxing 1; Liaoyan 241; Qiuguang; Yanfeng 47; Liaoyan 2; Tiejing 4; Liaojing 371 ; Liaojing 101) as the test material, planted in the greenhouse. The rice blast fungus ZA1, ZA9, ZB1 and ZF1 popular in Liaoning area were respectively transplanted on the oatmeal tomato juice agar medium, and cultured at 25-27°C for 5-7 days. After the aerial hyphae are removed and cultured 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 ...

Embodiment 3

[0053] Example 3: Using molecular marker InDel-1 to screen individuals carrying homozygous rice blast resistance gene Pi65(t) from hybrid offspring.

[0054] Using the rice blast resistance gene Pi65(t) donor variety Gangyu 129 as the male parent and the susceptible variety Liaoxing 1 as the female parent, F 1 F 2 Generation, F 2 The population is normally planted in the greenhouse, and each individual in the population is inoculated and identified according to the method described in Example 2 at the 5-leaf 1-heart stage, and the phenotype identification results are as follows: figure 2 shown. Extract F by the method described in Example 1 at the 6 leaf stage 2 The DNA of each individual plant of the generation population was amplified by PCR with primers of SEQ ID NO: 1 and SEQ ID NO: 2, and the amplification results were as follows: figure 2 shown. The results of inoculation identification and gene amplification showed that F 2 Individuals whose amplicon contained a...

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Abstract

The invention belongs to the field of molecular biology and particularly relates to a molecular marker assisted breeding method of a new rice variety with resistance to rice blast and special primers thereof. The molecular mark is a co-dominant molecular mark Indel-1 of the rice gene Pi65(t) with resistance to rice blast and is a nucleotide sequence which is amplified from total DNA of rice by using a primer pair SEQ ID NO: 1 and SEQ ID NO: 2. The method can be applied to molecular marker assisted selection of Pi65(t) in resistance breeding of the resistance to blast of rice, so that the efficiency of the anti-disease variety breeding is improved and the workload of field identification is reduced.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a molecular marker-assisted breeding method for a new blast-resistant rice variety and a special primer thereof. 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. Generally, it can cause a 10%-20% reduction in production, and when it occurs seriously, the loss can reach 40%-50% or even no harvest. Since the 1990s, the annual occurrence area of ​​rice blast in my country has been 3.8 million ha 2 More than hundreds of millions of kilograms of paddy is lost every year. Studies have shown that breeding disease-resistant rice varieties is the fundamental way to prevent and control rice blast, and the use of molecular markers of disease-resistant genes to assist in the sele...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11A01H1/02A01H1/04
CPCA01H1/02A01H1/04C12Q1/6895C12Q2600/156
Inventor 郑文静陆晓春赵家铭张丽霞马作斌丛玲王平白春明朱振兴王春语李丹李金红于惠琳王妍
Owner LIAONING ACAD OF AGRI SCI
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