A functional marker for detecting a broad-spectrum rice blast resistance gene PigmR and an application thereof

A rice blast resistance gene and function technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of easy loss of target genes, large genetic distance, etc. Long cycle and stable effect

Inactive Publication Date: 2019-01-11
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the markers for the PigmR gene that have been reported so far are all linked markers, and the genetic distance is relatively large, and the probability of gene exchange with PigmR is high, and the target gene is easily lost in the process of molecular marker-assisted selection breeding.

Method used

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  • A functional marker for detecting a broad-spectrum rice blast resistance gene PigmR and an application thereof
  • A functional marker for detecting a broad-spectrum rice blast resistance gene PigmR and an application thereof
  • A functional marker for detecting a broad-spectrum rice blast resistance gene PigmR and an application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: PigmR gene sequence analysis and design of functional markers

[0044] Currently, the molecular markers for identifying the PigmR gene are linkage markers. The present invention designs functional markers according to the functional sequence characteristics of the PigmR gene, so as to improve the identification effect and breeding application of the PigmR gene.

[0045] In Gumei No. 4 (from the Agricultural Germplasm Bank of Jiangsu Province), the Pigm gene cluster contains 13 R genes (R1-R13), among which R4, R6 and R8 can form complete transcripts. R6 is a disease resistance gene, which is a functional Pigm gene, called PigmR (sequence shown in SEQ ID NO.21), and there are no alleles in varieties or germplasms such as Nipponbare and 9311; R4 and PigmR genes are in protein There is a difference of 4 amino acids at the level, which has no function, and is called Pigm-R4 (sequence shown in SEQ ID NO.22); R8 is a susceptible gene, called PigmS (sequence shown i...

Embodiment 2

[0049] Example 2 Detection and optimization of functional markers

[0050] Genomic DNA from seedling leaves of Gumei 4, Nipponbare and Huanghuazhan was extracted by CTAB method (Murray M G, et al., Nucleic Acids Research, 1980, 8(19):4321-4326). Using the genomes of Gumei No. 4, Nipponbare and Huang Huazhan as templates, PCR screening was performed on the molecular markers for detecting the PigmR gene in Table 1. The PCR reaction system is 2 μL of genomic DNA, 2 μL of 10×PCR buffer, MgCl 2 (5mmol / L) 2μL, dNTP (2mmol / L) 2μL, upstream primer (2μmol / L) 2μL, downstream primer (2μmol / L) 2μL, Taq enzyme 0.2μL, ddH 2 O7.8 μL. The amplification conditions were 94°C for 5min; 35 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 30s; 72°C for 10min to end the reaction. PCR reactions were performed in an Eppendorf Mastercycle thermal cycler. Amplified products were separated by agarose gel electrophoresis and photographed in a gel imager, such as figure 1 shown. The results showed...

Embodiment 3

[0052] Example 3: Comparison of GMRA markers and Indel587 markers for detection of rice varieties

[0053]Indel587 is currently a conventional linkage marker for identifying the PigmR gene. The pigmR gene functional marker GMRA marker and the Indel587 marker developed by the inventors were used to detect and compare rice varieties. For the specific detection method, refer to the PCR detection method in Example 2 for detection. The PCR reaction system is 2 μL of rice plant genomic DNA, 2 μL of 10×PCR buffer, MgCl 2 2 μL, dNTP 2 μL, GMR-3F 4 μL, GMR-3R 4 μL, Actin1-1F 1 μL, Actin1-1R 1 μL, Taq enzyme 0.2 μL, ddH 2 O 1.8 μL. Amplification conditions were 94°C for 5 minutes; 94°C for 30s, 55°C for 30s, 72°C for 30s, 35 cycles; 72°C for 10 minutes, and the reaction was terminated. For the primer concentration, refer to Example 2 for the optimal concentration. The detection materials include indica rice plum No. 4, Nipponbare, Huang Huazhan, IR36, Yuzhenxiang, Nanjing No. 16, Kas...

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Abstract

The invention discloses a functional marker for detecting a broad-spectrum rice blast resistance gene PigmR and an application thereof. The marker includes a primer pair GMR-3 and/or prime pair Actin1-1, GMR-3 is designed according to the functional sequence of PigmR gene and completely co-separated with PigmR gene. The sample which could amplify PigmR gene contained 98 bp of PigmR gene and couldnot amplify the PigmR gene, which indicated that PigmR gene was not carried. Actin1-1. False negative results caused by the failure of PCR amplification of PigmR gene product in the process of excluding samples and experimental operation, the amplification product size is 146 bp. The marker and the method disclosed by the invention can be used for gene detection of variety resources and hybrid progeny, which is favorable for speeding up the breeding process of rice blast resistance. The method has the advantages of high accuracy, simple operation, high detection efficiency and low cost, and has strong practical value.

Description

technical field [0001] The invention belongs to the field of crop molecular breeding, and specifically relates to a functional marker for detecting a broad-spectrum rice blast resistance gene PigmR and an application thereof, in particular to a functional marker GMRA for detecting a PigmR gene derived from Gumei No. 4 and an application thereof. Background technique [0002] Rice blast is one of the main diseases that endanger rice production, and breeding rice varieties resistant to blast is the most effective way to solve the damage of rice blast. Traditional rice blast resistance genetic breeding is phenotypic identification by natural induction of rice blast or artificial inoculation of plants, which requires a lot of manpower and material resources, and is greatly affected by external factors, and the identification results often have large errors. Molecular marker-assisted selection is an effective way to improve the efficiency of rice breeding for blast resistance. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13
Inventor 王芳权杨杰许扬陈智慧王军李文奇范方军陈丽琴仲维功
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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