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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  • Abstract
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  • Claims
  • 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_442

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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[0058] Example 1. Design of primers for peanut high oleic acid related CAPS markers and PCR amplification to obtain homologous gene fragments

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

[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 sequence was compared and analyzed using GeneiousPro R11 software to find that these sequences It is clearly divided into 2 categories, namely AhFAD2A and AhFAD2B. The nucleotide sequences of these 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'end and 3'end, such as figure 1 As shown, the AhFAD2B gene has a 19 bp deletion base sequence 69 bp upstream of the start codon and a 15 bp deletion base seq...

Example Embodiment

[0089] Example 2. Restriction analysis of mutation sites

[0090] Look for suitable endonucleases for wild genotype or mutant genotype at the mutation site. The results showed that two different restriction enzyme digestion strategies can be adopted for different mutation sites, that is, C814T and G1111A mutations can find suitable restriction enzymes at the mutation sites of the mutant genotype. The expected result will be that of the wild-type individual. The amplified product cannot be cleaved by restriction enzymes. The amplified product of mutant strains can be cleaved by restriction enzymes, while the heterozygous genotype presents two band patterns; C301G and C313T mutations can be found in the wild genotype mutation sites. Enzyme, the expected result is that the amplified product of the wild-type single strain can be digested, the amplified product of the mutant single strain cannot be digested, and the heterozygous genotype presents two band types.

[0091] For 4 differen...

Example Embodiment

[0096] Example 3: Application of CAPS marker in the genotype identification of individual plants in the progeny population of high-oleic acid flower breeding species

[0097] The research material is randomly selected-200 F2 single plants of hybrid progeny of Jihua 6×C814T. As an exemplary example in the embodiment of the present invention, the gene mutation site-specific primer pair HOCAPS-F1 / HOCAPS-R1 and NdeI endonuclease, as well as the primer pair HOCAPS-F3 / HOCAPS-R3 and Hpy99I endonuclease respectively constitute a CPAS labeling system, using the F2 generation single plant genomic DNA as a template for specific PCR amplification, and then performing enzyme digestion reaction to obtain the band After the type, the genotype of the individual plant is analyzed and determined. The technical parameters and methods used are carried out in accordance with the above operations.

[0098] The results are shown in Table 2. The genetic law of AhFAD2A or AhFAD2B gene mutations in F2 popul...

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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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