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A functional molecular marker of rice blast resistance gene pi2 and its application

A resistance gene and molecular marker technology, applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve cumbersome steps, inability to distinguish Pi2 and Piz-t, multiple time and cost, etc. problem, achieve high specificity, improve breeding efficiency, and low cost

Active Publication Date: 2019-06-28
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, molecular markers for tracking rice blast resistance gene Pi2 generally use closely linked SSR markers such as AP22, which are a certain distance from Pi2, and the selection efficiency cannot reach 100%, and it cannot be directly used to detect whether the rice sample contains the Pi2 gene.
Using the sequence difference between the Pi2 gene sequence and the Nipponbare allele, a co-dominant molecular marker M-Pi2 of the Pi2 gene was established. Although this marker can identify Pi2, it cannot distinguish between Pi2 and Piz-t, and cannot identify pure hybrids. Synthetic genotype (Gao et al.2010)
Based on the molecular marker obtained by the single base difference of the Pi2 gene sequence, the product obtained by PCR amplification, after digestion with Pst I or Hinf I, was detected by polyacrylamide gel electrophoresis, and Pi2 can be specifically identified (Hua et al .2015, Tian et al.2016), but the steps of this method are relatively cumbersome, and it takes a lot of time and money in the process of industrialized molecular breeding

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  • A functional molecular marker of rice blast resistance gene pi2 and its application
  • A functional molecular marker of rice blast resistance gene pi2 and its application
  • A functional molecular marker of rice blast resistance gene pi2 and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0030]Example 1 Primer Design and Amplified Fragment Analysis of Rice Pi2 Gene Functional Markers 2Pi2-F and 2Pi2-R, 2WT-R / 2WT-1R

[0031] 1. Primer design

[0032] The rice database (http: / / www.gramene.org / ) and conventional PCR method were used to amplify and sequence Pi2 and its alleles of multiple rice varieties in the functional LRR (Leucine-Rich Repeats) region specificity of rice blast resistance Sexually conserved sequences, such as figure 1 within the dashed box shown; figure 2 It is the sequencing peak map of the BL122-Pi2 hybrid sequence in this region. It can be seen that the sequencing results of the nucleotides that are conservatively different from Pi2 and its alleles in this region are all double peaks; image 3 It is the nucleotide sequence comparison of the published Pi2, Pi9, Piz-t and other 21 varieties in this region (Tian et al.2016). It can be concluded that the conserved sequence of Pi2 in this region is: AGG A A TCTCAGAT GG Middle; the conserv...

Embodiment 2

[0043] Example 2 Identification of Pi2 in 9 Rice Resources Using Rice Pi2 Gene Functional Markers

[0044] 1. Extraction of rice genomic DNA

[0045] Using 11 rice varieties as materials: Guangqingzhan, Fengaozhan, Taixiaozhan, Qiguozhan, Jinkezhan, IR24, Zengcheng Simiao, Jinnongsimiao, Minghui 70, BL122-Pi2 hybrid and BL122 - Homozygous Pi2, numbered 1-11 in sequence. Using the CTAB method to extract rice genomic DNA, the specific steps are as follows: (1) Put a small amount of fresh rice leaves into a pre-cooled centrifuge tube, add liquid nitrogen and grind them into powder; (2) Add 600 μL of 65°C preheated CTAB lysis buffer solution, vortexed and mixed, and heated in a 65°C water bath for 45 minutes; (3) Add 600 μL of chloroform: isoamyl alcohol (volume ratio 24:1) mixture, turn over and mix, and centrifuge at 8,000 rpm for 10 minutes; (4) Transfer the supernatant to a new centrifuge tube, add 0.6 times the volume of isopropanol, invert and mix, let stand for 30min, 8,0...

Embodiment 3

[0050] Example 3 Correlation analysis between hybrid combination disease identification and Pi2 specific molecular marker detection

[0051] 1. Experimental materials and sources

[0052] The BL122 (Pi2 donor) co-plants B and Xiang B were crossed separately, which were recorded as the hybrid combination ① and ②; the CBB23 co-plant B and Xiang B were crossed separately, and were recorded as the hybrid combination ③ and ① and ③ (② and ) hybrid progenies were crossed in the F3 generation, and after the hybrids were selfed for 2 generations, rice blast resistance was identified in the F3 generation.

[0053] 2. The polymorphism of the functional molecular marker of rice blast resistance gene Pi2 among the four parents

[0054] Utilize the functional molecular marker of reported SSR primer AP22 and rice blast resistance gene Pi2 in the present invention to identify 4 parents, the PCR reaction system of each parent is: DNA template 1.0 μ L, primer (10 μ M 2Pi2-F 0.5 μL and 10 μ...

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Abstract

The invention discloses a functional molecular marker of a blast resistance gene Pi2 and application of the functional molecular marker. The functional molecular marker of the blast resistance gene Pi2 is characterized by comprising the following primers: 2Pi2-F: 5'-GACAGTTTCATTATGACAACTTTAG-3'; 2Pi2-R: 5'-AAGTGTTCCACCATCTGAGATTCCT-3'; 2WT-R: 5'-AAGTGTTCCAATATCTGATAATAAC-3'; or 2WT-1R: 5'-AAGTGTTCCAATAATACCTGAGAATAAC-3'. The functional molecular marker disclosed by the invention is simple, convenient and rapid and is low in cost; the functional molecular marker is applicable to assisted selection of molecular markers of segregation populations with rice blast resistance of Pi2 improved rice; and the breeding efficiency is improved and the requirements of large-scale molecular breeding are met.

Description

Technical field: [0001] The invention belongs to the field of crop molecular breeding, and in particular relates to a functional molecular marker of rice blast resistance gene Pi2 and its application. Background technique: [0002] my country is the country with the largest annual rice production in the world, and the stability of rice production is directly related to the national economy and people's livelihood. Rice blast caused by Magnaporthe grisea is one of the most serious diseases to rice production, known as the cancer of rice. At present, discovering and using broad-spectrum rice blast resistance genes to breed disease-resistant rice varieties is the main measure to control rice blast. Traditional rice disease-resistant breeding relies on artificial inoculation and selection of plant phenotypes, which is time-consuming and labor-intensive, and is affected by pathogen strains and pathogenic conditions, resulting in low breeding efficiency. Although the use of mole...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 张明永杨武范甜夏快飞曾璇胡锐邱迪洋李茂霖成太辉陈建通
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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