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Primers and method for detecting SNP gene typing of AhFAD2B genes of peanuts in high throughput

A genotyping and high-throughput technology, applied in the field of molecular biology, can solve the problem of high detection cost, achieve low detection cost, improve efficiency and accuracy

Inactive Publication Date: 2016-03-09
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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Problems solved by technology

[0004] Among the above detection methods, only the fluorescent quantitative PCR method can achieve high-throughput detection. The TaqMan probe method is very effective for the detection of InDel differences at 442 positions of the AhFAD2B gene. However, since the fluorescently labeled probes of this method are gene-specific probes, the detection cost is relatively higher

Method used

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  • Primers and method for detecting SNP gene typing of AhFAD2B genes of peanuts in high throughput
  • Primers and method for detecting SNP gene typing of AhFAD2B genes of peanuts in high throughput
  • Primers and method for detecting SNP gene typing of AhFAD2B genes of peanuts in high throughput

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Embodiment 1

[0033] Example 1. Design of primers for detection of AhFAD2B gene 442 A insertion mutation

[0034] The 442th base sequence of the ORF region of the AhFAD2B gene genome sequence has two sequences: wild type deletion and mutant type insertion of A base, resulting in the mutation of the encoded amino acid sequence from the wild type encoding a complete protein to the mutant type encoding a frameshift and premature termination. According to the position of the above-mentioned mutation site, design KASP allele differential primers (as shown in Table 1, SEQIDNo:1, SEQIDNo:2), so that the differential sequence of the AhFAD2B gene is located at the 3' end of the primer, and the 5' end is connected to the Allele -1tail and Allele-2tail, these two tailing sequences are identical to the sequences of the universal fluorescent probes in the LGC MasterMix detection kit, and the 5' ends of the probes are labeled with FAM and HEX fluorescent groups respectively (the probe sequence Patented b...

Embodiment 2

[0040] Embodiment 2, the detection of peanut AhFAD2B gene SNP mutation site

[0041] Allelic site detection was performed with primers designed according to the allelic difference of the AhFAD2B gene and LGC company kit MasterMix probe

[0042] Use the primers and MasterMix probes designed according to the allelic difference of the AhFAD2B gene in Example 2 to test peanut materials, including ordinary oleic acid peanuts (genotype aaBB) as the female parent and high oleic acid peanuts (genotype aabb) as the male parent. The F1 generation hybrids and the F2 generation segregation groups were detected, and the common oleic acid peanuts and high oleic acid peanuts of the female parents were used as controls at the same time. The specific methods are as follows:

[0043] (i) Extraction of genomic DNA from tested peanut samples

[0044] According to the Plant Genomic DNA Extraction Kit (catalogue number: DP-305) provided by TIANGEN Company, and according to the instructions of the ...

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Abstract

The invention relates to primers and a method for detecting SNP gene typing of AhFAD2B genes of peanuts in high throughput. The primers comprise the specific primer of which the nucleotide sequence is shown as SEQ ID No.1 and SEQ ID No.2 and a universal prime of which the nucleotide sequence is shown as SEQ ID No.3. According to the primers and method, the allelic variation condition of the AhFAD2B genes can be detected and identified in high throughput, the maximum sample quantity of one-time detection can reach 1536, the detection cost is low, rapidness and accuracy are achieved, and the efficiency and accuracy of peanut breeding selection are greatly improved.

Description

technical field [0001] The invention relates to a primer and a method for high-throughput detection of peanut AhFAD2B gene SNP genotyping, belonging to the technical field of molecular biology. Background technique [0002] Peanut is one of the four major oil crops in the world. The content and composition of fatty acid in its grain are important indicators to measure the quality of peanut. From the perspective of oil stability, peanuts and their products with high oleic acid / linoleic acid ratio (O / L ratio) are not easy to oxidative rancidity, and have a longer shelf life. Studies have shown that the storage life of roasted high-oleic peanuts is 8 times longer than that of ordinary peanuts with oleic acid content (Mozingo et al., 2004). Oleic acid is beneficial in maintaining levels of good cholesterol high-density lipoprotein (HDL), can enhance insulin sensitivity, and improve some inflammations (MesaGarcia et al., 2006; Vassiliou et al., 2009). In the industry, hydrogena...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/156C12Q2563/107
Inventor 单雷徐平丽唐桂英刘玮谷朝阳
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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