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United States Department of Agriculture

Agricultural Research Service

Research Project: CONSERVATION, CHARACTERIZATION, AND EVALUATION OF CROP GENETIC RESOURCES AND ASSOCIATED INFORMATION

Location: Plant Genetic Resources Conservation Unit

Title: A Simple Allele-Specific PCR Assay for Detecting FAD2 Alleles in Both A and B Genomes of the Cultivated Peanut for High Oleate Trait Selection

Authors
item Chen, Zhenbang -
item Wang, Ming
item Barkley, Noelle
item Pittman, Roy

Submitted to: Plant Molecular Biology Reporter
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 9, 2010
Publication Date: February 13, 2010
Repository URL: http://hdl.handle.net/10113/42005
Citation: Chen, Z., Wang, M.L., Barkley, N.L., Pittman, R.N. 2010. A Simple Allele-Specific PCR Assay for Detecting FAD2 Alleles in Both A and B Genomes of the Cultivated Peanut for High Oleate Trait Selection. Plant Molecular Biology Reporter. 28:542-548.

Interpretive Summary: Peanut seeds contain about 44-56% oil which is comprised of up to 12 fatty acids. Two major fatty acids oleic acid and linoleic acid account for approximately 80% of the oil composition. Peanut oils containing a high percentage of linoleic acid are prone to oxidation, leading to rancidity, off-flavors, and short shelf life during seed storage. Consuming oils with high levels of oleic acid is believed to beneficial to human health by reducing low density lipoproteins (LDL), maintaining high density lipoprotein (HDL), slowing down atherosclerosis, and reversing the inhibitory effect of insulin production. The conversion of oleic acid to linoleic acid is mainly catalyzed by the '12 fatty acid desaturase (FAD). Two homoeologous genes (FAD2A and FAD2B) encoding for the desaturase are located on the A and B genome, respectively. Abolishing or reducing the desaturase activity by gene mutation can significantly increase the oleic acid/linoleic acid ratio. Currently, detection of FAD2 alleles can be achieved by a cleaved amplified polymorphic sequence marker for the A genome and a real-time polymerase chain reaction (PCR) marker for the B genome; however, they have to be used with different assay platforms. A simple PCR assay for detection of FAD2 alleles on both genomes was developed by designing allele-specific primers and altering PCR annealing temperatures. This PCR assay is quick, reliable, economical, and easy to use. Implementation of this PCR assay will greatly enhance the efficiency of germplasm characterization and marker-assisted selection of high oleate in peanut.

Technical Abstract: In cultivated tetraploid peanut (2n = 4x = 40, AABB), the conversion of oleic acid to linoleic acid is mainly catalyzed by the '12 fatty acid desaturase (FAD). Two homoeologous genes (FAD2A and FAD2B) encoding for the desaturase are located on the A and B genome, respectively. Abolishing or reducing the desaturase activity by gene mutation can significantly increase the oleic acid/linoleic acid ratio. F435-derived high oleate peanut cultivars contain two key mutations within the '12 fatty acid desaturase gene which include a one base-pair substitution of G:C'A:T in the A genome and a one base-pair insertion of A:T in the B genome. Both mutations contribute to abolishing or reducing the desaturase activity leading to accumulation of oleate versus linoleate. Currently, detection of FAD2 alleles can be achieved by a cleaved amplified polymorphic sequence marker for the A genome and a real-time polymerase chain reaction (PCR) marker for the B genome; however, they have to be used with different assay platforms. A simple PCR assay for detection of FAD2 alleles on both genomes was developed by designing allele-specific primers and altering PCR annealing temperatures. This assay was successfully used for detecting FAD2 alleles in peanut. Gas chromatography (GC) was used to determine fatty acid composition of PCR assayed genotypes. The results from the PCR assay and GC analysis were consistent. This PCR assay is quick, reliable, economical, and easy to use. Implementation of this PCR assay will greatly enhance the efficiency of germplasm characterization and marker-assisted selection of high oleate in peanut.

Last Modified: 9/1/2014