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KASP marker typing primer combination for high-throughput detection of mutation sites of AhFAD2B gene and application of primer combination

A typing primer and high-throughput technology, applied in the field of molecular genetics and breeding, can solve the problem of blank KASP high-throughput typing technology, and achieve the effect of improving breeding efficiency, low cost, accurate and reliable results

Inactive Publication Date: 2019-04-09
INST OF CEREAL & OIL CROPS HEBEI ACAD OF AGRI & FORESTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the KASP high-throughput typing technology for the AhFAD2B-814 mutant gene is still blank

Method used

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  • KASP marker typing primer combination for high-throughput detection of mutation sites of AhFAD2B gene and application of primer combination
  • KASP marker typing primer combination for high-throughput detection of mutation sites of AhFAD2B gene and application of primer combination
  • KASP marker typing primer combination for high-throughput detection of mutation sites of AhFAD2B gene and application of primer combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Design of KASP marker and development of special primers for detection of C>T mutation site at position 814 of AhFAD2B gene in high oleic peanut C814T

[0050] The gene related to high oleic peanut oleic acid content involved in the present invention is AhFAD2B and its mutant gene AhFAD2B-814.

[0051] 1. Extraction of tested peanut material and its genomic DNA

[0052] 1. Selection of peanut materials for testing

[0053] The peanut materials for testing in the present invention are high oleic acid peanut new mutant C814T, high oleic peanut variety Jihua No. 16, and common oleic acid content peanut variety Jihua No. 5. The AhFAD2B genotypes of these peanut varieties have been reported (see for details Chinese patent document "A high oleic acid peanut mutant gene AhFAD2B-814 and its application", application number 201810774992.1), wherein the AhFAD2B genotype of C814T is bb (814C>T type), and the AhFAD2B genotype of Jihua 16 is b' b'(448G>A type, namely F43...

Embodiment 2

[0090] Example 2 Establishment of high-throughput KASP molecular marker detection method for AhFAD2B gene mutation site 814C>T in high oleic peanut C814T

[0091] 1. Genomic DNA extraction

[0092] For the extraction of genomic DNA from leaves of peanut varieties, refer to Step 1 of Example 1.

[0093] 2. PCR amplification reaction

[0094] Using the genomic DNA extracted in the above step 1 as a template, the KASP-labeled primers developed in Example 1 for detecting the peanut AhFAD2B gene were used for PCR amplification to obtain PCR amplification products.

[0095] KASP genotyping PCR reaction system (5 μL): including 2 μL template DNA (concentration about 20 ng / μL), primer mixture 0.07 μL, 2.5 μL 2×KASP Master Mix, 0.43 μL ddH 2 O.

[0096] Wherein, the final concentrations of the primers AhFAD2B-814F / C and AhFAD2B-814F / T in the primer mixture are both 12 μM, and the final concentration of the common primer AhFAD2B-814R / common is 25 μM. LGC's product 2×KASP Master Mix ...

Embodiment 3

[0105] Example 3 Application of high-throughput KASP molecular markers in the identification and breeding of high-oleic peanut germplasm

[0106] 1. Selection of peanut materials for testing

[0107] 1. The peanut germplasm materials for testing include: 16 peanut materials whose sequence information is known after the full-length sequencing of the AhFAD2B gene, as shown in Table 1, among which 10 materials have the AhFAD2B gene of the BB genotype: Yuanza 9102, Hua Yu 20, Ji 11-17-7-3, Ji 3-1-3, Ji Hua 20, Ji Hua 6, KX01-6, Ji Hua 11, Ji Hua 19, Ji Hua 13; AhFAD2B 5 copies of materials whose gene is bb genotype: C814T, 201508-412, 201508-957, 201521-602, 201511-023; 1 copy of materials whose gene AhFAD2B is Bb genotype: 201511-0481.

[0108] Table 1 Peanut materials used for high-throughput KASP molecular marker detection

[0109]

[0110] 2. The tested peanut crossbreeding and offspring include: using high oleic acid peanut mutant C814T to cross with ordinary oleic acid ...

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Abstract

The invention discloses a KASP marker typing primer combination for high-throughput detection of mutation sites of an AhFAD2B gene and an application of the primer combination, belongs to the technical field of molecular genetic breeding and relates to a special primer combination for detecting types of the AhFAD2B gene of high-oleic-acid peanuts by a high-throughput SNP molecular marker and the application of the primer combination. The primer combination is the KASP marker typing primer combination for detecting C>T mutant sites at 814 sites of the AhFAD2B gene of high-oleic-acid peanuts andcomprises an A-labeled wild type AhFAD2B gene specific primer with a nucleotide sequence shown in SEQ ID No.1, a B-labeled mutant AhFAD2B gene specific primer with a nucleotide sequence shown in SEQID No.2 as well as a AhFAD2B gene general primer with a nucleotide sequence shown in SEQ ID No.3. The high-throughput KASP molecular marker gene typing specific primers are designed for novel mutant 814 C>T allelic variation of the AhFAD2B of high-oleic-acid peanuts, novel mutants of the AhFAD2B can be effectively identified, and the problem that identifying methods in the prior art can only identify 448 G>A allelic variation of classic F435 mutant sites of the AhFAD2B is solved.

Description

technical field [0001] The invention belongs to the technical field of molecular genetic breeding, and relates to a combination of special primers for detecting the AhFAD2B genotype of high-oleic peanuts by using high-throughput SNP molecular markers and an application thereof. Background technique [0002] Peanut is an important source of edible vegetable oil and protein, and the content of oleic acid is an important quality index that affects the physical and chemical stability and nutritional value of peanut oil and peanut processed products. Oleic acid is a monounsaturated fatty acid, and the oleic acid molecule contains only one divalent unsaturated bond. Compared with polyunsaturated fatty acids (such as linoleic acid, etc.) containing two or more divalent unsaturated bonds, oleic acid The chemical stability of high oleic peanut oil is stronger, the high oleic acid peanut products are not easy to oxidative rancidity, and the shelf life is long. The storage period of hi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
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 INST OF CEREAL & OIL CROPS HEBEI ACAD OF AGRI & FORESTRY SCI
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