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Primer combination and method for detecting genotype of high oleic acid peanut and application

A primer combination and primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve the problems of cumbersome and complicated operations, high detection costs, and low accuracy, and achieve flexible throughput , good platform compatibility and high accuracy

Pending Publication Date: 2021-09-07
HEBEI UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the operations of CAPS, AS-PCR, gene sequencing, TaqMan probes and qRT-PCR technologies are cumbersome and complicated, requiring enzyme digestion, electrophoresis, sequencing, etc., and there are disadvantages such as low throughput and high detection cost; although KASP technology is a High-throughput allelic variation typing technology, but KASP requires Master Mix reagents for PCR amplification and a dedicated SNP typing detector, which is expensive. In addition, when typing complex target sequence sites There are also defects of low accuracy, and there is also the limitation that only fluorescence detection can be used

Method used

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  • Primer combination and method for detecting genotype of high oleic acid peanut and application
  • Primer combination and method for detecting genotype of high oleic acid peanut and application
  • Primer combination and method for detecting genotype of high oleic acid peanut and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1, the design of the functional gene marker STARP primer of peanut high oleic character

[0086] The genes involved in high oleic peanut oleic acid content were AhFAD2A and AhFAD2B and their mutant alleles AhFAD2Asnp and AhFAD2Bindel.

[0087] 1. Acquisition of allelic variant sequences of high oleic acid peanut functional genes AhFAD2A and AhFAD2B

[0088] From the literature "Expressed variants of Δ12-fatty acid desaturase for the highleate trait in spanish market-type peanut lines. López Y, Nadaf HL, Smith OD, Simpson C, Fritz A. (2004) Molecular Breeding, 9:183-192." AhFAD2A and AhFAD2B and their mutant alleles AhFAD2Asnp and AhFAD2Bindel were obtained from .

[0089] 2. Determine the mutation sites of the allelic variation of the AhFAD2A and AhFAD2B genes

[0090] The nucleotide sequences of the coding regions of the AhFAD2A and AhFAD2Asnp genes were compared using the MEGA-X software. The 448th base of the AhFAD2A gene is G, while the 448th base of th...

Embodiment 2

[0100] Example 2, Establishment of STARP Marking Method for High-Oleic Peanut Genotype Detection

[0101] 1. Extraction of tested peanut material and its genomic DNA

[0102] (1) Selection of peanut materials for testing

[0103] The peanut materials for testing in the present invention are high oleic acid peanut line 201731-1933 and common oleic acid content peanut line 201521-2612, wherein the AhFAD2A gene of 201731-1933 has G448A replacement, the genotype is AA homozygous type, and the AhFAD2B gene has 441_442InsA Mutation, the genotype is InsA homozygous type; AhFAD2A gene 448G and AhFAD2B gene 441_442 in 201521-2612 are not mutated, and the genotype is GG homozygous type and Del type respectively.

[0104] (2) Extraction of genomic DNA

[0105] A. Take a small amount of young peanut leaves (about 1g) and place them in a mortar, grind them into powder with liquid nitrogen, add 700 μL of 2% CTAB and 0.2β-mercaptoethanol extraction buffer that has been preheated in a 65°C ...

Embodiment 3

[0127] Example 3, Application of the method for identifying the FAD2 genotype by STARP markers in high oleic acid peanut breeding

[0128] Use the STARP marker developed by the present invention to detect the genotype of the RIL population family of the offspring of the hybrid combination of high oleic peanuts and ordinary peanuts, use the gene sequencing results to verify the typing results, test the fatty acid content of the RIL population family by gas chromatography, and analyze peanut oleic acid The relationship between content and genotype.

[0129] 1. Selection of peanut materials for testing

[0130] The peanut materials used in the test are 218 RIL population families of the offspring of the "0607-5×Wanhua No. 7" hybrid combination. The population has formed an F7 generation family through self-crossing and single-seed transmission. The internal genetics of the family are stable and the characters are uniform.

[0131] 2. Genomic DNA extraction

[0132] For extracti...

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Abstract

The invention relates to the technical field of biotechnology, and provides a primer combination and method for detecting genotype of high oleic acid peanut and application. The primer combination comprises a first primer group and a second primer group, wherein the first primer group is selected from any one or two of the primer group 1 and the primer group 3, the second primer group is selected from any one or two of the primer group 2 and the primer group 4, the primer group 1 and the primer group 3 are used for amplifying AhFAD2A, the primer group 2 and the primer group 4 are used for amplifying AhAFD2B, the primer group 1 comprises a specific primer group 1, the primer group 2 comprises a specific primer group 2, the specific primer group 1 comprises a primer 1, a primer 2 and a primer 3, the specific primer group 2 comprises a primer 4, a primer 5 and a primer 6, the primer group 3 comprises a primer 7, a primer 8 and the primer 3, and the primer group 4 comprises a primer 9, a primer 10 and the primer 6. The primer combination has the advantages of high throughput, low cost, high accuracy and suitability for multi-platform flexible operation.

Description

technical field [0001] The invention relates to the technical field of biotechnology, in particular to a primer combination, method and application for detecting genotype variation of high oleic peanuts. Background technique [0002] Peanut is one of the main oil crops and economic crops in my country. In recent years, the planting area of ​​peanuts in the country ranks second only to rapeseed among the bulk oil crops, and its total output accounts for more than half of the total domestic oil crops. Peanut has become the oil crop with the largest total output in my country. Among the bulk oil crops, peanut is the oil crop with the highest oil production efficiency. Half of the total peanut production is used for oil extraction. The annual peanut oil production accounts for more than a quarter of domestic vegetable oil production, and the proportion is steadily increasing, ranking first in domestic vegetable oil. two. The nutritional value and physical and chemical properti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/13C12Q2600/156C12Q2600/172C12Q2531/113C12Q2563/107C12Q2565/125
Inventor 陈四龙赵慧韩洁艾鹏飞郭学玲
Owner HEBEI UNIVERSITY OF SCIENCE AND TECHNOLOGY
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