Molecular marker method for detecting high oleic acid content of peanuts and application

A molecular marker, high oleic acid technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of limiting the wide and efficient application of new high-oleic acid peanut mutants, avoid phenotypic identification, Improve breeding efficiency and avoid time-consuming and labor-intensive effects

Active Publication Date: 2018-11-27
河北省农林科学院粮油作物研究所
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  • Application Information

AI Technical Summary

Problems solved by technology

Although CAPS marker technology has been applied to high oleic peanut germplasm identification and variety breeding at home and abroad, the marker methods used are based on the AhFAD2A G448A mutation and AhFAD2B 441_442insA mutation or MITE of the American high oleic peanut natural mutant F435. The development of high-oleic acid peanut mutants has limited the wide and efficient application of high-oleic acid peanuts in peanut breeding

Method used

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  • Molecular marker method for detecting high oleic acid content of peanuts and application
  • Molecular marker method for detecting high oleic acid content of peanuts and application
  • Molecular marker method for detecting high oleic acid content of peanuts and application

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Experimental program
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Effect test

Embodiment 1

[0058] Example 1, Peanut high oleic acid-related CAPS marker primer design and PCR amplification to obtain homologous gene fragments

[0059] 1.1 Sequence analysis and specific primer design of homologous genes AhFAD2A and FAD2B related to oleic acid content in peanut seeds

[0060] The sequence information of the peanut AhFAD2 gene was retrieved from the NCBI database (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov / ), and the obtained sequences were compared and analyzed using GeneiousPro R11 software, and it was found that these sequences Clearly divided into 2 categories, namely AhFAD2A and AhFAD2B. The nucleotide sequences of the two types of genes are highly similar, but there are multiple SNP difference sites in the coding region, and there are also obvious sequence differences at the 5' and 3' ends, such as figure 1 As shown, the AhFAD2B gene has a 19 bp missing base sequence at 69 bp upstream of the start codon and a 15 bp missing base seq...

Embodiment 2

[0089] Embodiment 2, enzyme digestion analysis of mutation site

[0090]Find the appropriate endonuclease for the wild type or mutant genotype at the mutation site. It was found that two different enzyme cutting strategies can be adopted for different mutation sites, that is, the C814T and G1111A mutations can find suitable restriction enzymes at the mutation site of the mutant genotype, and the expected result will be that of the wild-type single plant. The amplified product cannot be digested by enzymes, and the amplified product of a mutant individual plant can be digested by enzymes, while the heterozygous genotype presents two band patterns; C301G and C313T mutations can find suitable endonucleases at the mutation site of the wild genotype Enzyme, the expected result is that the amplified product of the wild-type individual plant can be digested, the amplified product of the mutant individual plant cannot be digested, and the heterozygous genotype presents two band patter...

Embodiment 3

[0096] Example 3: Application of CAPS markers in genotype identification of individual plants of high oleic peanut breeding progeny population

[0097] The research materials are 200 F2 single plants of the hybrid progeny of "Jihua No. 6 × C814T" selected randomly. NdeI endonuclease, and the primer pair HOCAPS-F3 / HOCAPS-R3 and Hpy99I endonuclease constituted the CPAS marker system respectively, using the above-mentioned F2 generation single plant genomic DNA as a template for specific PCR amplification, followed by enzyme digestion reaction to obtain bands After genotyping, single-plant genotype analysis and judgment were carried out. The technical parameters and methods used were all carried out in accordance with the above-mentioned operations.

[0098] The results are shown in Table 2, the mutation sites of the AhFAD2A or AhFAD2B genes in the F2 population genetic law alone present a segregation ratio of 1:2:1, which is in line with the Mendelian single gene inheritance law...

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Abstract

The invention discloses a molecular marker method for detecting high oleic acid content of peanuts and application and belongs to the technical field of molecular breeding of crops. The method comprises the following steps: aiming at mutation sites of nucleotide sequences of AhFAD2B genes with high oleic acid content of peanut seeds, amplifying and coding nucleotide sequence fragments of AhFAD2B genes by adopting primer pairs, and selecting corresponding restriction enzymes to perform digestion on amplification products, so that the mutation sites of the nucleotide sequences of the AhFAD2B genes with high oleic acid content of peanut seeds can be marked. According to the method disclosed by the invention, CAPS makers are successfully designed and developed for novel mutant genes of high-oleic-acid peanuts, and a kit for detecting the mutation sites is developed. The homozygous type and hybrid type of high oleic acid can be clearly identified, and genotyping can be performed in bred offspring segregation population, so that high oleic acid allelic individual plants or strains carrying out mutant genes are screened; and moreover, time- and labor-consuming phenotype identification canbe avoided, the breeding efficiency of high oleic acid peanut varieties is improved, and the breeding service life is shortened.

Description

technical field [0001] The invention belongs to the field of crop molecular breeding, and relates to a molecular marker method and application for detecting high oleic acid content in peanuts, in particular to a specific CAPS marker and primers that can be used to identify the wild type or mutant type of peanut high oleic acid gene AhFAD2B The pair and the application of the primer pair in the selection of high oleic peanut strains. Background technique [0002] Peanut is one of the important oil crops in the world, and it is also an important source of edible vegetable oil in my country. my country is the largest peanut production, consumption and trade exporter in the world. The fatty acid composition of peanuts is a key indicator to measure its quality. The oleic acid content of peanut seeds plays a decisive role in the physical and chemical stability and nutritional value of peanut products. Product shelf life and positive significance to human health, the oleic acid c...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 陈四龙刘义杰李玉荣程增书王瑾宋亚辉张朋娟
Owner 河北省农林科学院粮油作物研究所
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