Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A specific primer, molecular marker and detection method for efficiently detecting rice resistance gene pita

A technology of resistance genes and molecular markers, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. The effect of improving the detection efficiency and improving the breeding efficiency

Inactive Publication Date: 2019-02-12
YUNNAN AGRICULTURAL UNIVERSITY
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] In view of the above technical problems, the present invention intends to overcome the defects of false positives, time-consuming, expensive and not widely applicable in the existing detection technology.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A specific primer, molecular marker and detection method for efficiently detecting rice resistance gene pita
  • A specific primer, molecular marker and detection method for efficiently detecting rice resistance gene pita
  • A specific primer, molecular marker and detection method for efficiently detecting rice resistance gene pita

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Design and molecular markers of specific primers for efficient detection of rice resistance gene Pita key disease resistance loci

[0041] The primer design process is as follows: figure 1 As shown, the details are as follows:

[0042] (1) Sequence determination and mutation site analysis of the key disease resistance loci of the Pita gene:

[0043] 1. Seedling cultivation: the present invention selects Yunnan Dehong local rice varieties (137 Yunnan Dehong local rice varieties) to carry out seed collection, and seeds are sterilized in May (disinfection method: after 10% sodium hypochlorite solution disinfection for 5 minutes, use sterilized water Wash the seeds more than 10 times); then germinate in 30°C hydroponics in the incubator; transplant to the seedling pots in the greenhouse and use soil (mixture of paddy soil, humus, plant ash) as the substrate for planting for about 20 days (three days) Ye Yixin period);

[0044] 2. Sampling: Collect 142 rice seed...

Embodiment 2

[0067] Example 2 Detection of specific primer annealing temperature and specificity verification of Pita disease-resistant genotype

[0068] Annealing temperature is the most important factor affecting PCR specificity. After denaturation, the temperature is rapidly cooled to 40°C-60°C to allow primers and templates to combine. Since template DNA is much more complex than primers, the chances of collisional binding between primers and templates are much higher than those between complementary strands of templates. The annealing temperature and time depend on the length of the primer, its base composition and its concentration, and the length of the target base sequence.

[0069] By carrying out PCR amplification with the primers of the present invention and the primers of the prior invention at annealing temperatures of 53°C, 55°C, 58°C, 60°C, and 64.5°C respectively, the appropriate annealing temperature is screened, such as image 3 As shown in (A, B), the 1st and 18th lane...

Embodiment 3

[0074] Example 3 A method of using the molecular marker to detect key disease-resistant loci of the rice resistance gene Pita and large sample size verification

[0075] The method for using the molecular marker to detect the key disease resistance site of the rice resistance gene Pita comprises the following steps:

[0076] (1) Genomic DNA extraction of the rice plant of the sample to be tested;

[0077] (2) using the sample DNA obtained in step (1) as a template to construct a PCR reaction system;

[0078] (3) Utilize agarose gel electrophoresis to detect PCR amplification product, when PCR product is positive,

[0079] That is to say, a PCR product with a band length of 814bp was obtained, indicating that the resistance gene Pita 6640 site

[0080] The base is G, and the rice variety is resistant to blast fungus (including AVR-Pita); otherwise, when PCR produces

[0081] When the object is negative, that is, the PCR product with a band length of 814bp cannot be obtained,...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a specific primer for efficiently detecting key disease-resistant loci of rice resistance gene Pita, a molecular marker and a detecting method, and belongs to the field of rice genetic improvement and agricultural biological technology application. The specific primer consists of a forward primer (6640F) and a reverse primer (7415R), wherein the chain ratio of two variable nucleotide loci contained in the forward primer and the reverse primer in a large sample group reaches 99.31%, which shows that the primer has wide applicability to rice. According to a special PCR reaction system and reaction condition screened with the coordination of the primer designed by the invention, the accuracy and specificity of multiplication are greatly increased, and the problem of false positive of the primer used by predecessors in the detection process can be effectively solved. When the specific primer and the molecular marker which are designed by the invention are utilized to detect the disease-resistant / susceptible type of the rice gene Pita, only one-time PCR multiplication needs to be conducted, and the method has the advantages of being simple, fast and economical, and is suitable for quickly screening large samples and analyzing individuals and varieties with disease-resistant genes.

Description

technical field [0001] The invention relates to a specific primer, a molecular marker and a detection method for efficiently detecting key disease-resistant sites of rice resistance gene Pita, belonging to the fields of rice genetic improvement and agricultural biotechnology applications. technical background [0002] Rice blast is one of the main diseases that cause serious rice production reduction, and the production loss caused by rice blast accounts for about 10-15% of the total output every year. At present, discovering broad-spectrum rice blast resistance genes and cultivating rice varieties with broad-spectrum and long-lasting disease resistance through breeding are the main measures to control rice blast. Therefore, the detection of rice blast resistance genes and the screening of blast resistance rice germplasm are particularly important. [0003] Molecular markers related to disease resistance genes are of great significance in genetic breeding. Marker-assisted s...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13
Inventor 刘立娜李成云杨静张寒
Owner YUNNAN AGRICULTURAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com