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A primer set for identifying rice blast fungus and its screening method and application

A rice blast fungus and primer set technology, applied in biochemical equipment and methods, genomics, proteomics, etc., can solve problems such as time-consuming, labor-intensive, heavy workload, and limited throughput

Active Publication Date: 2022-02-25
HUNAN HYBRID RICE RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Magnaporthe grisea can be identified by field inoculation method, among which, complete gene resequencing needs to isolate Magnaporthe grisea, which is also time-consuming and laborious. At the same time, the amount of sequencing is too large, resulting in insufficient sequencing depth, which leads to a decrease in accuracy; SSR marker method in PCR (Polymerase Chain Reaction, Polymerase Chain Reaction) During the amplification process, there are phenomena such as slippage, which will lead to low detection accuracy and sensitivity. SSR markers also need to be amplified and detected one by one, and the throughput is limited. This leads to a heavy workload; the SNP marker polymorphism in the SNP marker method is low, and the method of whole-genotype resequencing is mostly used to detect SNP markers. Therefore, the defects of whole-gene resequencing also exist in the SNP marker method, and some use The SNP chip is used for detection, which has the problem that the chip technology error is more than 1%. The chip design is complex and a large number of chips need to be synthesized and tested at one time to reduce the unit cost, resulting in a high overall cost for each use and the inability to flexibly adjust anything that needs to be detected. marker site

Method used

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  • A primer set for identifying rice blast fungus and its screening method and application
  • A primer set for identifying rice blast fungus and its screening method and application
  • A primer set for identifying rice blast fungus and its screening method and application

Examples

Experimental program
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Embodiment 1

[0040] The screening method of primer group, comprises the following steps:

[0041] 1) Use the 300 genome sequence databases of Magnaporthe grisea and use the formatdb command in the BLAST toolkit provided by the National Center for Biotechnology Information (NCBI) to construct an index file for the database. Select the most complete genome sequence assembled as the query sequence, use BLASTN software (version number is 2.9.0) to compare the query sequence with the genome sequence database of 300 Magnaporthe grisea, and the output format is -outfmt 1, And set the maximum number of output sequences to 300, and align all genome sequences in the genome sequence database neatly.

[0042]2) Translate along the neatly aligned genome sequence with a window of 125 bp, and move 5 bp each time, and perform a pairwise comparison of 300 genome fragments of Magnaporthe grisea within the window for each translation, if two fragments If there are more than 2 SNPs, it is considered to be di...

Embodiment 2

[0054] Two different monoclonal sources of rice blast fungus were isolated from two different rice areas in Hunan, numbered 1 and 2 respectively, and No. 1 rice blast fungus was divided into two parts, numbered 1- 1 and 1-2. Therefore, No. 1-1 and No. 2 are different rice blast fungus, No. 1-2 and No. 2 are rice blast fungus, and No. 1-1 and 1-2 are the same rice blast fungus.

[0055] The application of the primer set in the identification of rice blast bacterium comprises the following steps:

[0056] S1. Using the primer set to amplify the detection sites of the genomic DNA of the rice blast fungus to be tested and the control blast fungus oryzae by multiplex PCR to obtain multiplex PCR amplification products.

[0057] In this embodiment, all primer sets from site 1 to site 100 in Table 1 were synthesized and mixed in Thermoelectric Corporation of the United States. The primer set includes a forward primer and a reverse primer. The sequence of the primer set is as SEQ ID N...

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Abstract

The invention relates to a primer set for identifying rice blast fungus and its screening method and application. The primer set includes a forward primer and a reverse primer, and the sequences of multiple primers in the primer set are shown in SEQ ID NO: 1 to SEQ ID NO: 200 in the sequence listing. The screening method of the primer set includes: 1) using the genome sequence of Magnaporthe grisea as a query sequence, and comparing it with a genome sequence database, and aligning all genome sequences neatly; 2) aligning all genome sequences neatly; Do translation; every time translation is performed pairwise comparison of multiple genome fragments of Magnaporthe grisea in the window, leaving discriminative gene fragments as candidate marker sites; 3) for each candidate marker site between multiple genomes 4) Screen out the primer set. The primer set can identify rice blast fungus accurately and at low cost. The present invention also includes the use of the above primer sets.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a primer set for identifying rice blast fungus and a screening method and application thereof. Background technique [0002] Rice blast is one of the most important diseases of rice, causing a large amount of grain loss every year, and its pathogen is Magnaporthe grisea. The traditional identification method of rice blast fungus is field inoculation, which is very time-consuming and laborious. DNA molecular identification methods mainly include whole gene resequencing, SSR (Simple Sequence Repeat) marking method and SNP (Single Nucleotide Polymorphism) marking method identification. Magnaporthe grisea can be identified by field inoculation method, among which, complete gene resequencing needs to isolate Magnaporthe grisea, which is also time-consuming and laborious. At the same time, the amount of sequencing is too large, resulting in insufficient sequencing depth, which lead...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11G16B20/30G16B25/20
CPCC12Q1/6895C12Q1/686G16B20/30G16B25/20C12Q2537/143
Inventor 邢俊杰彭海邓华凤彭志荣石银丰
Owner HUNAN HYBRID RICE RES CENT