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A modified PCR protocol for consistent amplification of fatty acid desaturase (FAD) alleles in marker-assisted backcross breeding for high oleic trait in peanut

Qiao, L., Pandey, M., Anglin, N., Feng, S., Wang, H., Culbreath, A., Varshney, R.ORCID: 0000-0002-4562-9131 and Guo, B. (2013) A modified PCR protocol for consistent amplification of fatty acid desaturase (FAD) alleles in marker-assisted backcross breeding for high oleic trait in peanut. In: 6th International Peanut Conference (AAGB 2013), 17 - 21 June 2013, Zhengzhou, China.

Abstract

High oleic acid, such as is found in olive oil, is desirable for the healthy cholesterol-lowering benefits. The oxidative stability of the oil with high oleic acid also gives longer “shelve life” for peanut products. These benefits drive the breeding effort toward developing high oleic peanuts worldwide. Two homoeologous genes (FAD2A and FAD2B) encode the fatty acid desaturase (FAD) which regulates the conversion of oleic acid to linoleic acid. There have been several published protocols that can track the mutant alleles of FAD2 genes, including: CAPS, real-time PCR, and a simple allele-specific PCR assay. The objective of this research is to develop a user-friendly PCR-based protocol that could be used in any PCR machine for marker-assisted breeding coupled with an accelerated backcross program. These genetic markers can identify true hybrids in backcrossing, and the backcross and selection process can be accelerated by using heterozygous hybrids as a donor. In three years of laboratory work plus several years of field testing, a high oleic elite cultivar could be generated in contrast to conventional breeding, which takes over 10 years for a new cultivar release. In this study, a modified protocol has been developed and used in four different PCR machines successfully. This simple and cost-effective method could be used to track FAD2A and FAD2B alleles of heterozygous genotypes in backcross breeding selection. This improved protocol is not PCR cycler-specific, but PCR condition-specific. Consistent amplification was achieved using different PCR machines each with modified PCR conditions, and these results were further confirmed using real-time PCR assay. Thus, both the FAD alleles present on A (FAD2A) and B (FAD2B) genomes can be detected and tracked in rapidly and economically for a breeding program.

Item Type: Conference Item
URI: http://researchrepository.murdoch.edu.au/id/eprint/63134
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